Caveolae and its structural protein caveolin-1 (Cav-1) are abundant in vascular endothelial cells (ECs) and have been suggested to contribute to cell signaling and cholesterol trafficking. This study investigated the effect of cholesterol on the movement of caveolae-related proteins in human umbilical vein ECs with use of caveolae functional proteomics. After cholesterol exposure to ECs for 2 to 4 h, caveolae were isolated and separated on 2-D protein gels. Among 40 protein spots revealed in caveolae fractions, the ATP synthase beta subunit (ATPS-beta), one of the 3 proteins enriched by cholesterol in caveolae, was confirmed by western blotting and confocal microscopy. Further, cholesterol exposure increased the level of ATPS-beta, along with Cav-1 and cholesterol in caveolae. These effects could be blocked by cytochalasin B, an actin cytoskeleton disruptor. ATPS-beta was physically associated with Cav-1, as demonstrated by co-immunoprecipitation and GST-Cav-1 fusion protein pull-down assay. Cholesterol increased the extracellular ATP release mediated by ATPS-beta, since this action could be blocked by piceatannol or oligomycin, ATPS inhibitors. Thus, the ectopic localization of ATPS-beta may participate in the energy balance of cells in response to the change in intracellular cholesterol levels.