After Anfinsen earned his Ph.D. from Harvard Medical School in 1943, the university hired him to teach courses in biochemistry. In 1944, the Office of Scientific Research and Development (OSRD), a newly established wartime bureau created during Franklin D. Roosevelt's administration by scientific visionary Vannevar Bush, sponsored a research project at Harvard to study malaria. The OSRD wanted to understand the biological makeup of the malarial parasite Plasmodium knowlesi, which is transmitted by mosquitoes, in order to protect U.S. troops stationed in Southeast Asia, North Africa, and the South Pacific. The OSRD recruited Anfinsen as a civilian biochemist, and he spent the next three years, until 1946, studying the metabolism of blood in both healthy monkeys and monkeys infected with malarial parasites.
After 1946, Anfinsen resumed teaching at Harvard Medical School, where he eventually became an assistant professor of biochemistry. Over the next decade, his research shifted from examining the metabolic processes of tissues to focussing on biochemical processes at the molecular level. This shift in focus was part of a growing scientific trend, as many of Anfinsen's peers and colleagues in the late 1940s were making the transition from cellular to molecular biochemistry. Anfinsen became increasingly interested in proteins. He investigated the catalytic proteins known as enzymes, and the smaller molecules known as amino acids that, when linked together in chain-like combinations, comprise proteins. Working with Harvard biochemist Arthur K. Solomon, Anfinsen used radioactive isotopes to examine the activity of amino acid chains. In 1947, the American Cancer Society sponsored Anfinsen as a senior fellow for a year at the Medical Nobel Institute in Stockholm, where he worked with the noted biochemist Hugo Theorell.
In 1950, James Shannon, associate director in charge of research at the National Heart Institute, part of the National Institutes of Health, invited Anfinsen to become Chief of the Laboratory of Cellular Physiology. Many of Anfinsen's colleagues were surprised by his move from the prestige of Cambridge to a federal position in Bethesda. But, as Anfinsen recalled in 1985, "It was hard to turn down this offer, partly because of its scientific potential, and also because the move would double my salary overnight." Over the course of the next three decades, Anfinsen's various laboratories in Bethesda would sponsor an astonishing array of talented postdoctoral and staff researchers including future NIH director Donald S. Fredrickson, future Nobel Laureate Martin Rodbell, and Michael Sela, future director of the Weizmann Institute of Science in Israel. In fact, the Weizmann Institute invited Anfinsen as a Guggenheim Fellow for a year in 1958-59, and in 1962 invited him to serve on its Board of Governors. Anfinsen held this position at Weizmann for almost thirty-five years.