<jats:title>Abstract</jats:title><jats:p>The purpose of the present study was to clarify roles of cytosolic chloride ion (<jats:styled-content style="fixed-case"><jats:roman>Cl</jats:roman></jats:styled-content><jats:sup>−</jats:sup>) in regulation of lysosomal acidification [intra‐lysosomal pH (pH<jats:sub>lys</jats:sub>)] and autophagy function in human gastric cancer cell line (<jats:styled-content style="fixed-case">MKN</jats:styled-content>28). The <jats:styled-content style="fixed-case">MKN</jats:styled-content>28 cells cultured under a low <jats:styled-content style="fixed-case"><jats:roman>Cl</jats:roman></jats:styled-content><jats:sup>−</jats:sup> condition elevated pH<jats:sub>lys</jats:sub> and reduced the intra‐lysosomal <jats:styled-content style="fixed-case"><jats:roman>Cl</jats:roman></jats:styled-content><jats:sup>−</jats:sup> concentration ([<jats:styled-content style="fixed-case"><jats:roman>Cl</jats:roman></jats:styled-content><jats:sup>−</jats:sup>]<jats:sub>lys</jats:sub>) <jats:italic>via</jats:italic> reduction of cytosolic <jats:styled-content style="fixed-case"><jats:roman>Cl</jats:roman></jats:styled-content><jats:sup>−</jats:sup> concentration ([<jats:styled-content style="fixed-case"><jats:roman>Cl</jats:roman></jats:styled-content><jats:sup>−</jats:sup>]<jats:sub>c</jats:sub>), showing abnormal accumulation of <jats:styled-content style="fixed-case">LC</jats:styled-content>3II and p62 participating in autophagy function (dysfunction of autophagy) accompanied by inhibition of cell proliferation <jats:italic>via</jats:italic> G<jats:sub>0</jats:sub>/G<jats:sub>1</jats:sub> arrest without induction of apoptosis. We also studied effects of direct modification of <jats:styled-content style="fixed-case"><jats:roman>H</jats:roman></jats:styled-content><jats:sup>+</jats:sup> transport on lysosomal acidification and autophagy. Application of bafilomycin A1 (an inhibitor of V‐type <jats:styled-content style="fixed-case"><jats:roman>H</jats:roman></jats:styled-content><jats:sup>+</jats:sup>‐<jats:styled-content style="fixed-case">ATP</jats:styled-content>ase) or ethyl isopropyl amiloride [<jats:styled-content style="fixed-case">EIPA</jats:styled-content>; an inhibitor of <jats:styled-content style="fixed-case"><jats:roman>Na</jats:roman></jats:styled-content><jats:sup>+</jats:sup>/<jats:styled-content style="fixed-case"><jats:roman>H</jats:roman></jats:styled-content><jats:sup>+</jats:sup> exchanger (<jats:styled-content style="fixed-case">NHE</jats:styled-content>)] elevated pH<jats:sub>lys</jats:sub> and decreased [<jats:styled-content style="fixed-case"><jats:roman>Cl</jats:roman></jats:styled-content><jats:sup>−</jats:sup>]<jats:sub>lys</jats:sub> associated with inhibition of cell proliferation <jats:italic>via</jats:italic> induction of G<jats:sub>0</jats:sub>/G<jats:sub>1</jats:sub> arrest similar to the culture under a low <jats:styled-content style="fixed-case"><jats:roman>Cl</jats:roman></jats:styled-content><jats:sup>−</jats:sup> condition. However, unlike low <jats:styled-content style="fixed-case"><jats:roman>Cl</jats:roman></jats:styled-content><jats:sup>−</jats:sup> condition, application of the compound, bafilomycin A1 or <jats:styled-content style="fixed-case">EIPA</jats:styled-content>, induced apoptosis associated with increases in caspase 3 and 9 without large reduction in [<jats:styled-content style="fixed-case"><jats:roman>Cl</jats:roman></jats:styled-content><jats:sup>−</jats:sup>]<jats:sub>c</jats:sub> compared with low <jats:styled-content style="fixed-case"><jats:roman>Cl</jats:roman></jats:styled-content><jats:sup>−</jats:sup> condition. These observations suggest that the lowered [<jats:styled-content style="fixed-case"><jats:roman>Cl</jats:roman></jats:styled-content><jats:sup>−</jats:sup>]<jats:sub>c</jats:sub> primarily causes dysfunction of autophagy without apoptosis <jats:italic>via</jats:italic> dysfunction of lysosome induced by disturbance of intra‐lysosomal acidification. This is the first study showing that cytosolic <jats:styled-content style="fixed-case"><jats:roman>Cl</jats:roman></jats:styled-content><jats:sup>−</jats:sup> is a key factor of lysosome acidification and autophagy.</jats:p>