The daughter of a postman, Lonsdale (née Yardley) was born at Newbridge in Ireland and moved to England with her family in 1908. She studied physics at Bedford College, London, graduating in 1922, and spent most of the following 20 years based at the Royal Institution in the research team of William Henry Bragg. In 1946 she moved to University College, London, where she served as professor of chemistry and head of the department of crystallography from 1948 until her retirement in 1968.

Lonsdale was one of the early pioneers of x-ray crystallography, centered on the Royal Institution and the team headed by Bragg and including such scholars as William Astbury, John Bernal, Dorothy Hodgkin, and John Robertson. It was from this group that most of the concepts and techniques of the new discipline emerged in the 1920s and 1930s. Lonsdale herself was responsible for one of the first demonstrations of the power of the new techniques when, in 1929, she published details of the structure of benzene. Working with a large crystal of hexamethylbenzene she established the hexagonal nature of the ring, that it was planar to within 0.1 angstrom (Å = 10^–10 m), and that the carbon–carbon bonds were 1.42 Å. This was followed in 1931 by the equally significant structure of the more difficult hexachlorobenzene, the first investigation of an organic compound in which Fourier analysis was used.

Other crystallographic subjects researched by Lonsdale included the magnetic susceptibility of crystals and the structure of synthetic diamonds and, in the 1960s, that of bladder stones. She edited the first three volumes of the International Tables for X-ray Crystallography (1952, 1959, 1962) and also produced a survey of the subject in her Crystals and X-rays (1948).

As a Quaker and a convinced pacifist Lonsdale refused to register in 1939 for government service or civil defense despite the fact that as a mother of three young children she would have been exempted from any such service. Fined £2 in 1943, she refused to pay and served a month in Holloway prison instead.

When, 285 years after its foundation, the Royal Society finally decided to admit women to its fellowship Lonsdale was the first to be elected (1945) and she became the society's vice-president in 1960. She was appointed a Dame of the British Empire in 1956 and also became, in 1968, the first woman to serve as president of the British Association for the Advancement of Science.