After graduating in medicine from the University of Rome in 1896—the first woman in Italy to do so—Montessori was appointed assistant doctor at the psychiatric clinic of the University of Rome, where she became interested in the educational problems of mentally retarded children. Between 1899 and 1901 she served as director of the State Orthophrenic School of Rome, where her methods proved extremely successful. From 1896 to 1906 she held a chair in hygiene at a women's college in Rome, and from 1900 to 1907 she lectured in pedagogy at the University of Rome, holding a chair in anthropology from 1904 to 1908. During these years she continued her studies of philosophy, psychology, and education.

In 1907 Montessori opened the first Casa dei Bambini (“Children's House”), a preschool for children ages three to six from the San Lorenzo slum district of Rome, applying her methods now to children of normal intelligence. Her successes led to the opening of other Montessori schools, and for the next 40 years she travelled throughout Europe, India, and the United States lecturing, writing, and establishing teacher-training programs. In 1922 she was appointed government inspector of schools in Italy, but left the country in 1934 because of the Fascist rule. After periods in Spain and Ceylon (now Sri Lanka), she settled in The Netherlands.

Montessori scorned conventional classrooms, where “children, like butterflies mounted on pins, are fastened each to his place.” She sought, instead, to teach children by supplying concrete materials and organizing situations conducive to learning with these materials.

She discovered that certain simple materials aroused in young children an interest and attention not previously thought possible. These materials included beads arranged in graduated-number units for premathematics instruction; small slabs of wood designed to train the eye in left-to-right reading movements; and graduated series of cylinders for small-muscle training. Children between three and six years old would work spontaneously with these materials, indifferent to distraction, for from a quarter of an hour to an hour. At the end of such a period, they would not seem tired, as after an enforced effort, but refreshed and calm. Undisciplined children became settled through such voluntary work. The materials used were designed specifically to encourage individual rather than cooperative effort. Group activity occurred in connection with shared housekeeping chores.

A large measure of individual initiative and self-direction characterized the Montessori philosophy, and self-education was the keynote of the plan. The teacher provided and demonstrated the special “didactic apparatus” but remained in the background, leaving the child to handle it for himself. In the Montessori system biological and mental growth are linked. “Periods of sensitivity,” corresponding to certain ages, exist when a child's interest and mental capacity are best suited to the acquisition of certain specialized knowledge.

Montessori's methods are set forth in such books as Il metodo della pedagogia scientifica (1909; The Montessori Method, 1912), The Advanced Montessori Method (1917–18), The Secret of Childhood (1936), Education for a New World (1946), To Educate the Human Potential (1948), and La mente assorbente (1949; The Absorbent Mind, 1949).