Bittner was born in Meadville, Pennsylvania. He studied biology and gained his doctorate at the University of Michigan in 1930 for original research on tumours arising in hybrid mice. This was an important start to his subsequent career — John Bittner spend the greater part of his academic life involved in cancer research. He was George Chase Christian Professor of Cancer Research and Director of the Division of Cancer Biology at the University of Minnesota's medical school (1943–61).

While working at the Jackson Laboratory in Bar Harbor, Maine (1936), Bittner made the important discovery that some strains of mice were highly resistant to cancer, while others were prone to it. Also, if the young of cancer-resistant mice were transferred to cancer-prone mothers they became cancerous, apparently via the mothers' milk, whereas cancer-resistant parents induced resistance in cancer-prone young. The discovery of viruslike organisms in the milk of cancer-prone parents suggested that these organisms are the cause of the cancer. Bittner called this a “milk factor”. Bittner's findings followed, and may be linked with those of Francis Rous, who made the controversial finding that other virus like organisms are, perhaps, the cause of sarcomas (tumours originating in connective tissue) in chickens.

Bittner authored, or co-authored, over 240 papers on cancer research and was extremely influential and important in the general scientific approach to cancer research.