SIRT1 plays a key role in maintaining metabolic homeostasis in mammals by directly modulating the activities of various transcription factors and metabolic enzymes through lysine deacetylation. White adipose tissue plays a key role in lipid storage and metabolism. To identify novel molecular targets of SIRT1 in fat cells, we used a non-biased proteomic approach. We identified a number of proteins whose acetylation status was significantly affected by SIRT1 modulator treatment in 3T3-L1 adipocytes. Among them, ATP6V1B2, a subunit of the vacuolar (H+)-ATPase, was further shown to be associated with SIRT1 by co-immunoprecipitation assay. Moreover, SIRT1 deacetylates ATP6V1B2 in vitro and in vivo. Taken together, our study demonstrates that ATP6V1B2 is a molecular target of SIRT1 in fat cells and the role of SIRT1 and ATP6V1B2 acetylation in the vacuolar (H+)-ATPase function warrants further investigation.