Recent studies show that ion channels/transporters play important roles in fundamental cellular functions. Several reports indicating the important roles of Cl- channels/transporters on cell proliferation suggest that the intracellular chloride concentration ([Cl-]i) regulated by them would be one of critical messengers. We investigated whether the [Cl-]i controls cell proliferation and cell cycle progression in human gastric cancer cells. Our studies indicated that furosemide, a blocker of Na+ /K+ /2Cl- cotransporter (NKCC), diminished cell growth by delaying the G1-S phase progression in gastric cancer cells with high expression and activity of NKCC. Furthermore, we found that the culture in the low Cl- medium (replacement of Cl- by NO3-) decreased the [Cl-]i and inhibited cell growth of gastric cancer cells and that this inhibition of cell growth was due to cell cycle arrest at the G0/G1 phase caused by diminutionof CDK2 and phosphorylated Rb. The culture of cells in the low Cl- medium significantly increased expressions of p21 mRNA and protein. In addition, the low Cl- medium induced phosphorylation of mitogen activated protein kinases (MAPKs). Treatment with an inhibitor of p38 or JNK significantly suppressed p21 upregulation caused by culture in a low Cl- medium and rescued gastric cancer cells from the low Cl- -induced G1 cell cycle arrest. These findings revealed that the [Cl-]i affects the cell proliferation via activation of MAPKs through upregulation of p21 in gastric cancer cells. Our results suggest that the [Cl-]i regulates important cellular functions in gastric cancer cells, leading to the development of novel therapeutic strategies. Recent reports show that ion channels / transporters play important roles in fundamental cellular functions. Several reports indicating the important roles of Cl- channels / transporters on cell proliferation suggest that the intracellular chloride concentration ([Cl-] i) regulated by them would be one of critical messengers. We investigated whether the [Cl-] i controls cell proliferation and cell cycle progression in human gastric cancer cells. Our studies indicated that furosemide, a blocker of Na + / K + / 2Cl- cotransporter (NKCC), diminished cell growth by Further, we found that the culture in the low Cl-medium (replacement of Cl- by NO3-) decreased the [Cl-] i and inhibited cell growth of gastric cancer cells and that this inhibition of cell growth was due to cell cycle arrest at the G0 / G1 phase caused by diminution of CDK2 and phosphorylated Rb. The culture of cells in the low Cl- med In addition, the low Cl-medium induced phosphorylation of mitogen activated protein kinases (MAPKs). Treatment with an inhibitor of p38 or JNK significantly suppressed p21 upregulation caused by culture in a low Cl- medium These findings revealed that the [Cl-] i affects the cell proliferation via activation of MAPKs through upregulation of p21 in gastric cancer cells. Our results suggest that the [ Cl-] i regulates important cellular functions in gastric cancer cells, leading to the development of novel therapeutic strategies.