Inhibition of heat shock protein 90 (Hsp90) has emerged as a novel intervention for the treatment of solid tumors and leukemias. Here, we report that F(1)F(0)-ATP synthase, the enzyme responsible for the mitochondrial production of ATP, is a co-chaperone of Hsp90. F(1)F(0)-ATP synthase co-immunoprecipitates with Hsp90 and Hsp90-client proteins in cell lysates of MCF-7, T47D, MDA-MB-453, and HT-29 cancer cells. Inhibition of F(1)F(0)-ATP synthase by efrapeptins results in the disruption of the Hsp90 complexing with its substrate proteins and, in most cases, in the degradation of the latter. Hsp90-client proteins affected by the inhibition of F(1)F(0)-ATP synthase included ERalpha, mutated p53 (m.p53), Hsp70, Hsp27, and caspase-3 but not Raf-1. This is the first report identifying caspase-3 as a substrate protein of Hsp90. Unlike typical Hsp90 inhibitors, efrapeptin treatment triggers Hsp70 downregulation in parallel with depletion of Hsp90. This suggests that suppression of Hsp90 chaperone function through inhibition of F(1)F(0)-ATP synthase does not result in activation of transcription factor HSF-1, a generally unfavorable consequence of anti-cancer treatments based on Hsp90 inhibition.