Acid-electrolyzed water (AEW) is commonly used as a disinfectant in the agricultural and medical fields. Although several studies have been conducted to examine its toxicity in vitro and in vivo, the cytotoxic mechanism of AEW has never been verified. The purpose of the present study was to elucidate the underlying mechanism by which AEW exerts its in vitro cytotoxic effect. Mouse fibroblasts treated with AEW experienced dilution rate-dependent cytotoxic effects in the 100% confluent phase as well as in the mitotic phase. The levels of intracellular reactive oxygen species (ROS) increased significantly in fully-confluent cells treated with undiluted and four times diluted AEW. In both of these treatments, cytotoxicity was also observed. It is thus concluded that the in vitro cytotoxicity of AEW is attributable to increased intracellular ROS. Additionally, the ROS responsible for these effects appears to be, at least in part, hydroxyl radical because the increase in intracellular ROS was attenuated by post-treatment with dimethyl sulfoxide, a hydroxyl radical scavenger, and with the antioxidant polyphenol, proanthocyanidin.