LASS2/TMSG1 could interact with the C subunit of vacuolar ATPase (Ⅴ-ATPase, ATP6V0C) and regulate Ⅴ-ATPase activity.In an attempt to provide molecular mechanism of the interaction between LASS2/TMSG 1 and Ⅴ-ATPase, we constructed four variant transfectants containing different functional domain of LASS2/TMSG1 and stably transfected the variants to human prostate cancer cell line PC-3M-1E8 cell with high metastatic potential.Results showed Ⅴ-ATPase activity and Intracellular pH (pHi) was significantly higher in the variant transfectants with Homeodomain of LASS2/TMSG1 than that in the control Immunoprecipitation, immunofluorescence and immunoelectron microscope alone or in combination demonstrated the direct interaction of Homeodomain of LASS2/TMSG1 and ATP6V0C.Loss of Homeodomain markedly enhanced the proliferation ability but weakened the apoptotic effect of LASS2/TMSG1 in PC-3M-1E8 cells.LASS2/TMSG1 could regulate Ⅴ-ATPase activity and intracellular pH through the direct interaction of its Homeodomain and the C subunit of V-ATPase.Their interaction could play impotant roles in the apoptosis of tumor cells.