The regulation of the luminal pH of each organelle is crucial for its function and must be controlled tightly. Nevertheless, it has been assumed that the nuclear pH is regulated by the cytoplasmic proton transporters via the diffusion of H⁺ across the nuclear pores because of their large diameter. However, it has been demonstrated that ion gradients exist between cytosol and nucleus suggesting that the permeability of ions across the nuclear pores is restricted. V-H⁺-ATPase is responsible for the creation and maintenance of trans-membrane electrochemical gradient. We hypothesize that V-H⁺-ATPase located in the nuclear membranes functions as the primary mechanism to regulate nuclear pH and generate H⁺ gradients across the nuclear envelope. We studied the sub-cellular heterogeneity of H⁺ concentration in the nucleus and cytosol using ratio imaging microscopy and SNARF-1, a pH indicator, in prostate cells. Our results indicated that there are proton gradients across the nuclear membranes that are generated by V-H⁺-ATPase located in the outer and inner nuclear membranes. We demonstrated that these gradients are mostly dissipated by inhibiting V-H⁺-ATPase. Immunoblots and V-H⁺-ATPase activities corroborated the existence of V-H⁺-ATPase in the nuclear membranes. This study demonstrates that V-H⁺-ATPase is functionally expressed in nuclear membranes and is responsible for nuclear H⁺ gradients that may promote not only the coupled transport of substrates, but also most electrochemically driven events across the nuclear membranes. This study represents a paradigm shift that the nucleus can regulate its own pH micro-environment, providing new insights into nuclear ion homeostasis and signaling.