Insulin-producing cells express limited activities of anti-oxidative enzymes. Therefore, reactive oxygen species (ROS) produced in these cells play a crucial role in cytotoxic effects. Furthermore, diabetes mellitus (DM) development is closely linked to higher ROS levels in insulin-producing cells. Hita Tenryosui Water® (Hita T. W., Hita, Japan) and Nordenau water (Nord. W., Nordenau, Germany), referred to as natural reduced waters (NRWs), scavenge ROS in cultured cells, and therefore, might be a possibility as an alternative to conventional pharmacological agents against DM. Therefore, this study aimed to investigate the role of NRWs in alloxan (ALX)-induced β-cell apoptosis as well as in ALX-induced diabetic mice. NRWs equally suppressed DNA fragmentation levels. Hita T. W. and Nord. W. ameliorated ALX-induced sub-G1 phase production from approximately 40% of control levels to 8.5 and 11.8%, respectively. NRWs restored serum insulin levels (p < 0.01) and reduced blood glucose levels (p < 0.01) in ALX-induced mice. Hita T. W. restored tissue superoxide dismutase (SOD) (p < 0.05) activity but not tissue catalase activity. Hita T. W. did not elevate SOD or catalase activity in HIT-T15 cells. Nord. W. restored SOD (p < 0.05) and catalase (p < 0.05) activity in both cultured cells and pancreatic tissue to normal levels. Even though variable efficacies were observed between Hita T. W. and Nord. W., both waters suppressed ALX-induced DM development in CD-1 male mice by administering NRWs for 8 weeks. Our results suggest that Hita T. W. and Nord. W. protect against ALX-induced β-cell apoptosis, and prevent the development of ALX-induced DM in experimental animals by regulating ALX-derived ROS generation and elevating anti-oxidative enzymes. Therefore, the two NRWs tested here are promising candidates for the prevention of DM development.