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Objective: To reveal the importance of histidine in intestinal epithelial cells, we investigated whether reduction in histidine concentration affects cell viability and apoptosis in rat intestinal epithelial cells (IEC-6). Methods: Culture cells were incubated in DMEM with or without histidine. Cell viability was measured by the MTT assay. The expression of caspase-3, -8, -9, and -12 was evaluated by western blot analysis. Activated caspase-3 was used as an apoptosis index. To determine the apoptosis pathway, activated caspase-8, -9, and -12 were also examined. Mitochondrial dysfunction was evaluated by the mitochondrial membrane potential assay. Some experiments were also performed on other cells (gastric mucosal cells or kidney cells), to compare with the results of IEC-6 cells. Results: Histidine deficiency significantly reduced cell viability after 6 h and induced caspase-3-dependent apoptosis after 9 h, in IEC-6 cells. Also, histidine deficiency decreased the mitochondrial membrane potential after 6 h. Therefore, we speculated that apoptosis was induced by mitochondrial dysfunction. Additionally, histidine at concentrations higher than 10 μM prevented the decrease in cell viability and the facilitation of apoptosis in IEC-6 cells. In rat gastric mucosal cells, similar results to the IEC-6 histidine deficiency results were obtained, but not in rat kidney cells. Conclusions: This is the first report showing that histidine deficiency reduced cell viability and induced apoptosis in IEC-6 cells, and that a small amount of histidine supplementation prevented and improved the IEC-6 cell injury. This is a potential new clinical treatment against intestinal and/or gastric cell injury that would improve the patient's quality of life.