Rotblat was educated at the University of Warsaw. In 1939 he was appointed to a research fellowship at the University of Liverpool to work on neutron fission in the laboratory of James Chadwick. Like many other Europeans, he worked during World War II at Los Alamos on the development of the atom bomb. But unlike most other scientists as soon as it was clear in early 1945 that Germany was defeated and would be unable to produce a nuclear weapon in time, Rotblat felt unable to justify working any longer on the development of such weapons. He was also alarmed by the attitude of some of his senior colleagues. General Groves, for example, the head of the Manhattan Project, was overheard by Rotblat insisting that the real purpose of the bomb was to subdue Soviet Russia.

Consequently he resigned from Los Alamos, arousing the deep suspicion of the security officers who suspected him of being a Soviet agent, and returned to Liverpool in early 1945. He remained at Liverpool until 1950 when he was appointed professor of physics at Bart's Hospital Medical College, a position he held until his retirement in 1976. Rotblat's scientific work was mainly concerned with radiation medicine.

He did, however, concern himself with other matters. After the explosion of the H-bomb on Bikini atoll, the Atomic Energy Commission reported that the fallout from this and all other explosions was no more damaging to the individual than the exposure received from a single diagnostic x-ray. The announcement did nothing to reassure Rotblat. The Bikini bomb, he worked out independently, would have been much more dangerous than the authorities admitted. Further, he reasoned, chest x-rays screen only chests; nuclear bombs radiated the whole body including wombs, ovaries, and testicles.

Rotblat was determined not to let the matter rest. He drafted an appeal for peace, backed by Bertrand Russell and Albert Einstein among others, to be delivered to world leaders. He realized, however, that something more permanent and constructive was needed. Consequently he set about raising money to hold a series of conferences in which technical matters could be debated, authoritative proposals made, and contacts established between scientists from different disciplines and countries.

The American millionaire Cyrus Eaton offered to finance the first conference as long as it was held in the Nova Scotian fishing village of Pugwash, Eaton's birthplace. The first conference was held in 1957 and was attended by 22 scientists. Known as Pugwash conferences, ever since they have continued to be held around the world. Rotblat served as the Secretary-General of Pugwash from its inception until 1973. Since 1988 he has held the office of Pugwash President.

Rotblat was awarded the 1995 Nobel Peace Prize, an award he shared with the organization he helped to found, the Pugwash Conferences on Science and World Affairs. The award was made, the Nobel committee announced, to mark the 50th anniversary of Hiroshima and Nagasaki and to protest against French nuclear tests in the Pacific.