Since phenoxazine is an essential structure of actinomycin D, which exerts a strong anticancer effect, we examined the anticancer effect of 2-aminophenoxazine-3-one (Phx-3) on mouse malignant melanoma B16 cells in vitro and in vivo. Phx-3 inhibited proliferation of the B16 cells in a dose-dependent manner in vitro. We furthermore studied the in vivo effects of Phx-3 on mouse malignant melanoma B16 cells transplanted in female C57BL/6Cr Slc mice. Treatment with Phx-3 (0.5 mg/kg) completely suppressed the growth of mouse malignant melanoma B16 cells transplanted in mice as compared with the control group. Phx-3 was found to exert few adverse effects, in terms of bodyweight loss, changes in serum levels of blood biochemical parameters such as aspartate transaminase (AST), alanine transaminase (ALT), blood urea nitrogen (BUN) and creatinine, dysfunction of the liver and the kidney examined by pathological methods, piloerection and wasting, when mice were treated with a dose of 0.5 mg/kg. These results suggest that Phx-3 may be used to treat patients affected by malignant melanoma in future.