Inhibition of oxidative phosphorylation (OXPHOS) is a promising therapeutic strategy for select cancers that are dependent on aerobic metabolism. Here, we report the discovery, optimization, and structure-activity relationship (SAR) study of a series of novel OXPHOS inhibitors. The hit compound, benzene-1,4-disulfonamide 1, was discovered in a phenotypic screen selective for cytotoxicity in a galactose-containing medium. Our multi-parameter optimization campaign led to the discovery of 65 (DX3-235), showing nanomolar inhibition of complex I function and adenosine triphosphate (ATP) production in a galactose-containing medium resulting in significant cytotoxicity. Importantly, 64 (DX3-234), a close analogue of 65, is well tolerated in mice and shows significant single agent efficacy in a Pan02 syngeneic pancreatic cancer model, suggesting that highly potent and selective OXPHOS inhibitors can be useful for the treatment of pancreatic cancer.