Nirenberg earned his B.S. (1948) in zoology and chemistry and his M.S. (1952) in zoology at the University of Florida. He received his Ph.D. in biological chemistry from the University of Michigan in 1957 and that year joined the staff of the National Institutes of Health (NIH) in Bethesda, Md. His research earned him the National Medal of Science in 1965, and the following year he was elevated to director of biochemical genetics at the NIH, a position he held for the remainder of his career. In 1968 Nirenberg and Khorana were recognized with an Albert Lasker Basic Medical Research Award and the Louisa Gross Horowitz Prize for Biology or Biochemistry.

In the late 1960s Nirenberg's research shifted from genetics to neurobiology. He began investigating neuroblastomas—tumours involving masses of neurons, known as ganglia—and eventually developed a neuroblastoma model that served as the basis for a broad range of neurobiological research. In the 1970s Nirenberg used his model as a platform for explorations into morphine's effects on the nervous system and neural synapse formation in chicken retinas. During this time scientists discovered that under the influence of certain factors normal genes could be “switched on,” becoming overactive in the form of oncogenes (cancer-causing genes). This finding, which demonstrated that gene activity could change and that these changes could affect cell growth, stimulated Nirenberg's interest. His research had begun to focus on nervous system growth and development, but how these processes were controlled was unknown. Nirenberg reasoned that to further understand the development of the nervous system, it was necessary to understand the genes that had the greatest influence on neurological development in the embryo. By the late 1980s a set of genes, known as homeobox genes (discovered in 1983), had become central to his studies. His experiments concerning homeobox genes and the assembly of the nervous system in Drosophila (fruit fly) were crucial to the advancement of the field of neurobiology. Much of Nirenberg's work on nervous system development in Drosophila proved relevant to studies on the development of the nervous system in humans.