The present study was attempted to evaluate the effects of gamma-irradiated doxorubicin (IRD) on spleen cell proliferation, cytokines release (IFN-gamma and IL-2) and lung metastasis in mice. Gamma irradiation induced degradation of doxorubicin molecule and cytotoxicity on melanoma (B16BL6) and myoblast (H9c2) cell lines were determined by high performance liquid chromatography (HPLC) and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide tetrazole) assay, respectively. Non-irradiated doxorubicin (NIRD) was used as a control. The mice injected with NIRD (2mg/kg body weight for 5 days, 24h interval) showed a considerable decrease (P<0.05) in the body, spleen weight, proliferation and cytokine release (IL-2 and IFN-gamma) as compared to control. However, a non-significant variation was observed in IRD treated mice compared with normal. Tumor bearing mice treated with NIRD and IRD (2mg/kg body weight, five doses at 48 h interval) showed diverse results on spleen cell cytokine release, proliferation and metastasis. HPLC results revealed the formation of several trace level degradation (P<0.05) products of IRD. IRD displayed a non-significant variation of cytotoxicity on B16BL6 cells, and low percentage (P<0.01) of cardiotoxicity on H9c2 cells as compared to NIRD. Altogether, this present study emphasis that gamma irradiation altered the property of doxorubicin.