Objective: Promotion of the proliferative expansion of CD4 Foxp3 regulatory T cells (Tregs) is one of the side effects that limits the use of bleomycin (BLM) in the treatment of tumors. In this study, we examined the hypothesis that cyclophosphamide (CY), a chemotherapeutic agent with the capacity to eliminate tumor infiltrating Tregs, abrogated BLM-induced expansion of Tregs and consequently resulted in a better anti-tumor effect. Methods: The in vitro effects of BLM, with or without mafosfamide (MAF, the active metabolite of CY), on both TGF-®-induced differentiation of Tregs (iTregs), and TNF-induced expansion of naturally occurring Tregs (nTregs) were assessed. The in vivo effect of low doses of BLM and CY on tumor-infiltrating Tregs, as well as on the growth of mouse B16-F10 melanomas, was also studied. Results: In vitro treatment with BLM promoted the differentiation of iTregs, as well as TNF-induced expansion of nTregs. These effects of BLM were completely abrogated by MAF. Furthermore, in the mouse B16-F10 melanoma model, treatment with low doses of BLM increased the number of tumor-infiltrating Tregs, and this effect of BLM was also abrogated by CY. Importantly, combination therapy with low doses of BLM and CY showed synergistic anti-tumor effects. Conclusions: CY abrogated the effect of BLM on the expansion of Tregs. The combination of these 2 chemotherapeutic agents may represent a safer and more effective therapy in the treatment of cancer patients, and thus merits future clinical evaluation.