Negative ions are considered to have potential health benefits, but few studies have examined their effects in vivo. We studied water-generated negative ions (WNI) with respect to physical properties as well as immunologic activation and anti-tumor activity (inhibition of carcinogenesis and tumor growth) in mice. Electrically, generated negative ions (ENI) served as control. Water-generated negative ions had a long life, significantly enhanced the cytotoxic activity of natural killer cells, and significantly decreased the incidence of cancer and inhibited tumor growth. Anti-tumor effects were attributed to enhancement of natural killer cell activity. The mechanisms and applications of negative ions warrant further investigation.