[4] The novel centers around a colony of escaped lab rats—the rats of NIMH—who live in a technologically sophisticated and literate society mimicking that of humans.

They come to the aid of Mrs. Frisby, a widowed field mouse who seeks to protect her children and home from destruction by a farmer's plow.

[7] Mrs. Frisby, a recently widowed mouse, lives with her four children in a cinder block in a field belonging to a farmer named Mr. Fitzgibbon.

An older mouse named Mr. Ages, who was a friend of Mrs. Frisby's late husband Jonathan, gives her some medicine for Timothy.

They have technology such as elevators, have tapped into the electrical grid to provide lighting and heating, and have acquired other human skills, such as storing food for the winter.

While captured, Frisby overhears the farmer and his family discussing an incident at a nearby hardware store in which a group of rats was electrocuted after seemingly attempting to steal a small motor.

[3] In 1985, Alethea K Helbig called Mrs. Frisby "a combination of science fiction and animal fantasy" that described "fantastic situations with scientific accuracy".

[8] Scholar Paula T. Connolly noted the book for Conly's "gradations of moral understanding and culpability" while dealing with "such problematic issues as the roles of science and technology, identity, idealism, family life, forms of community and means of survival".

[9] In a retrospective essay about the Newbery Medal–winning books from 1966 to 1975, children's author John Rowe Townsend wrote: "It seems to me that the fact that all the animals talk and behave intelligently from the beginning of the story detracts from the spectacular development of the laboratory rats... Mrs. Frisby and the Rats of NIMH is a pleasing book, but I find it mildly frustrating; it might have been something more than it is".

[8] A culture of fear began to grow surrounding unethical medical and scientific practices, which are heavily reflected in the book.

Constance Vidor commented that "Conly's books continue her father's emphasis on the theme of social responsibility while weaving in new characters with more personal problems".

The film adds a mystical element completely absent from the novel, with Nicodemus portrayed as a wise, bearded old wizard with magic powers and an enchanted amulet, rather than as an equal of the other rats.

[13] In 2009, Paramount Pictures set Neil Burger to write the script and Cary Granat to produce the film based on the book.