Vacuolar ATPase (V-ATPase), widespread in eukaryotic cells, is extensively expressed in many highly metastatic tumors, of which the V-ATPase c subunit ATP6V0C is particularly associated with the invasion and metastasis of cancer. ATP6V0C was directly found to interact with LASS2/TMSG1 which is a new tumor metastasis inhibitory gene identified by our laboratory in 1999. In order to study the role of ATP6V0C, we generated small interference RNA (siRNA) targeting ATP6V0C and investigated its function on the invasion of human prostate cancer cell line PC-3M-1E8 with high metastatic potential and its interplay with LASS2/TMSG1. We found that the expression of ATP6V0C was higher in prostate cancer cell lines PC-3M-1E8 and PC-3M with high metastatic potential than that from cell lines PC-3M-2B4 and PC-3 with low metastatic potential, indicating that ATP6V0C enhanced metastatic capacity in prostate cancer cells. Furthermore, silencing of ATP6V0C in PC-3M-1E8 cells inhibited V-ATPase activity (by ~5-fold), decreased extracellular hydrogen ion concentration and successively decreased activation of secreted MMP-9 (by ~3.6-fold), which coincided with the inhibition of cell migration and invasion in vitro, as well as a marked decrease in the expression of LASS2/TMSG1 probably through positive feedback. Thus we concluded that silencing of the ATP6V0C gene effectively suppressed the migration and invasion of prostate carcinoma cells through the inhibition of the function of V-ATPase, not through a LASS2/TMSG1-dependent manner. Therefore ATP6V0C inhibitors are promising therapeutic targets for advanced prostate cancer.