It is known that, besides its direct cytotoxic effect as an alkylating chemotherapeutic agent, cyclophosphamide also has immuno-modulatory effects, such as depletion of CD4+CD25+ regulatory T cells. However, its optimal concentration has not yet been fully elucidated. Therefore, we first compared the effects of different doses of cyclophosphamide on T cell subsets including CD4+CD25+ T cells in mice. Cyclophosphamide (20 mg/kg) decreased the numbers of splenocytes, CD4+ and CD8+ T cells by approximately 50%, while a decline in CD4+CD25+ T cell number was more profound, leading to the remarkably lower ratios of CD4+CD25+ T cells to CD4+ T cells. In contrast, 200 mg/kg cyclophosphamide severely decreased the numbers of all the T cell subsets by > 90% although the decreased ratios of CD4+CD25+ T cells to CD4+ T cells were still observed. Next, low-dose cyclophosphamide significantly inhibited in vivo growth of murine hepatoma MH129 tumor in immuno-competent but not immuno-deficient mice. This anti-tumor effect was abolished by CD4+CD25+ T cell repletion. In contrast, high-dose cyclophosphamide exhibited similar anti-tumor effects in both mice. In addition, contrary to antibody-mediated CD4+CD25+ T cell depletion, administration of low-dose cyclophosphamide after tumor inoculation was more efficacious than the prior administration. Our data show that low-dose cyclophosphamide selectively depletes CD4+CD25+ T cells, leading to enhanced anti-tumor effects against pre-existing tumors, while the anti-tumor effect of high-dose cyclophosphamide is solely attributed to its direct cytotoxicity. These findings appear to be highly crucial in a clinical setting of combined chemotherapy and immunotherapy for cancer treatment.