Biphenyl is a universal intermediate agent used as a protectant in various industrial activities. Biphenyl is currently used in postharvest applications as a fungicide in foreign countries to maintain the safety and quality of agricultural products. However, the risk of using biphenyl is in dispute in Japan. The toxicity of biphenyl has been studied in animals, and reportedly affects the liver and kidney especially. However, the toxic effect of biphenyl on cells is currently not well-understood, and the mechanism is unclear. We examined the toxicity of biphenyl on HL60 cells, a human promyelocytic leukemia cell line, by performing flow cytometry analyses with fluorescent probes. Biphenyl at 100 μM or greater significantly increased lethality and the intensity of side scatter on HL60 cells. Moreover, biphenyl at 30 μM or greater increased intracellular Ca2+ in a concentration-dependent manner. This increase resulted from free extracellular Ca2+ entering into the cells. These results indicate that an increase in intracellular Ca2+ may be one of several causes of the cytotoxicity induced by biphenyl. This study will contribute to the safety evaluation of biphenyl in the future.