Dorothea Brooke is an idealistic, well-to-do young woman, engaged in schemes to help the lot of the local poor. She is seemingly set for a comfortable, idle life as the wife of neighbouring landowner Sir James Chettam, but to the dismay of her sister, Celia (who later marries Chettam), and of her loquacious uncle Mr. Brooke, she marries instead Edward Casaubon, a middle-aged pedantic scholar who, she believes, is engaged on a great work, The Key to All Mythologies. She wishes to find fulfilment through sharing her husband’s intellectual life, but during an unhappy honeymoon in Rome she experiences his coldness towards her ambitions. Slowly she realises that his great project is doomed to failure and her feelings for him descend to pity. She forms a warm friendship with a young cousin of Casaubon’s, Will Ladislaw, but her husband’s antipathy towards him is clear (partly based on his belief that Ladislaw is trying to seduce Dorothea in order to gain access to Casaubon's fortune) and Ladislaw is forbidden to visit. In poor health, Casaubon attempts to extract from Dorothea a promise that, should he die, she will "avoid doing what I should deprecate and apply yourself to do what I desire" — meaning either that she should shun Ladislaw, or, as Dorothea believes, that she will complete The Key to All Mythologies in his place. Before Dorothea can give her reply, Casaubon dies. She then learns that he has added the extraordinary provision to his will that, if she should marry Ladislaw, Dorothea will lose her inheritance from Casaubon.

Meanwhile, an idealistic young doctor, Tertius Lydgate, has arrived in Middlemarch, with advanced ideas for medical reform. His voluntary hospital work brings him into contact with the town’s financier, Mr. Bulstrode, who has philanthropic leanings, but who is also a religious zealot with a secret past. Bulstrode's niece is Rosamond Vincy, the mayor's daughter and the town's recognised beauty, who sets her sights on Lydgate, attracted by what she believes to be his aristocratic connections and his novelty. She wins him, but the disjunction between her self-centredness and his idealism ensures that their marriage is unhappy. Lydgate manages his financial affairs badly and he is soon deep in debt and has to seek help from Bulstrode. He is partly sustained emotionally in his marital and financial woes by his friendship with Camden Farebrother, the generous-spirited and engaging parson from a local parish.

At the same time we have become acquainted with Rosamond's university-educated, restless, and somewhat irresponsible brother, Fred, reluctantly destined for the Church. He is in love with his childhood sweetheart, Mary Garth, a sensible and forthright young woman, who will not accept him until he abandons the Church (which she knows he has no interest in) and settles in a more suitable career. Mary has been the unwitting cause of Fred’s loss of a considerable fortune, bequeathed to him by the aged and irascible Mr. Featherstone, then rescinded by a later will which Featherstone, on his death-bed, begs Mary to destroy. Mary, unaware of what is at stake, refuses to do so. Fred, in trouble over some injudicious horse-dealing, is forced to borrow from Mary’s father, Caleb Garth, to meet his commitments. This humiliation shocks Fred into a reassessment of his life and he resolves to train as a land agent under the forgiving Caleb.

These three interwoven narratives, with side-plots such as the disastrous though comedic attempt by Mr. Brooke to enter Parliament as a sponsor of Reform, are the basis of the story until it is well into its final third. Then a new thread emerges, with the appearance of John Raffles, who knows about Bulstrode’s past and is determined to exploit this knowledge. Bulstrode’s terror of public exposure as a hypocrite leads him to hasten the death of the mortally sick Raffles by giving him access to forbidden alcohol and excess amounts of opium. But he is too late; Raffles had already spread the word. Bulstrode’s disgrace engulfs the luckless Lydgate, as knowledge of the financier’s loan to the doctor becomes public, and he is assumed to be complicit with Bulstrode. Only Dorothea and Farebrother maintain faith in Lydgate, but Lydgate and Rosamond are threatened by the general opprobrium with the necessity of leaving Middlemarch. The disgraced and reviled Bulstrode’s only consolation is that his wife stands by him as he, too, faces exile.

The final thread in the complex weave concerns Ladislaw, who, since their initial meeting, has kept his love for Dorothea to himself. He has remained in Middlemarch, working for Mr. Brooke, and has also become a focus for Rosamond’s treacherous attentions. After Brooke’s election campaign collapses, there is nothing to keep Ladislaw and he visits Dorothea to make his farewell. But Dorothea, released from life with Casaubon, but still the prisoner of his will, now sees Ladislaw as the means of her escape to a new life. Renouncing her independence, and Casaubon's fortune, she shocks her family again, by announcing she will marry Ladislaw. At the same time Fred, who has proved an apt pupil in Caleb’s profession, finally wins the approval and hand of Mary.

Beyond the principal stories we are given constant glimpses into other scenes. We observe Featherstone’s avaricious relatives gathering for the spoils, visit Farebrother’s strange ménage, and become aware of enormous social and economic divides. But these are the backdrops for the main stories which, true to life, are left largely suspended, leaving a short finale to summarise the fortunes of our protagonists over the next thirty years or so. The book ends as it began, with Dorothea: "Her full nature [ . . . ] spent itself in channels which had no great name on the Earth. But the effect of her being on those around her was incalculably diffusive: for the growing good of the world is partly dependent on unhistoric acts."