ABSTRACT: The purpose of the present study was to clarify roles of cytosolic chloride ion (Cl) in regulation of lysosomal acidification [intra-lysosomal pH (pHlys)] and autophagy function in human gastric cancer cell line (MKN28). The MKN28 cells cultured under a low Cl condition elevated pHlys and reduced the intra-lysosomal Cl concentration ([Cl]lys) via reduction of cytosolic Cl concentration ([Cl]c), showing abnormal accumulation of LC3II and p62 participating in autophagy function (dysfunction of autophagy) accompanied by inhibition of cell proliferation via G0/G1 arrest without induction of apoptosis. We also studied effects of direct modification of H transport on lysosomal acidification and autophagy. Application of bafilomycin A1 (an inhibitor of V-type H-ATPase) or ethyl isopropyl amiloride [EIPA; an inhibitor of Na/H exchanger (NHE)] elevated pHlys and decreased [Cl]lys associated with inhibition of cell proliferation via induction of G0/G1 arrest similar to the culture under a low Cl condition. However, unlike low Cl condition, application of the compound, bafilomycin A1 or EIPA, induced apoptosis associated with increases in caspase 3 and 9 without large reduction in [Cl]c compared with low Cl condition. These observations suggest that the lowered [Cl]c primarily causes dysfunction of autophagy without apoptosis via dysfunction of lysosome induced by disturbance of intra-lysosomal acidification. This is the first study showing that cytosolic Cl is a key factor of lysosome acidification and autophagy.