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 <b>1</b>, was discovered in a phenotypic screen selective for cytotoxicity in a galactose-containing medium. Our multi-parameter optimization campaign led to the discovery of <b>65</b> (<b>DX3-235</b>), showing nanomolar inhibition of complex I function and adenosine triphosphate (ATP) production in a galactose-containing medium resulting in significant cytotoxicity. Importantly, <b>64</b> (<b>DX3-234</b>), a close analogue of <b>65</b>, 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.