Cytotoxic lymphodepletion therapies augment antitumor immune responses. The generation and therapeutic efficacy of antitumor effector T cells (T_Es) are enhanced during recovery from lymphopenia. Although the effects of lymphodepletion on naive T cells (T_Ns) and T_Es have been studied extensively, the influence of lymphodepletion on suppressor cells remains poorly understood. In this study, we demonstrate a significant increase of CD4⁺CD25⁺Foxp3⁺ regulatory T cells (Tregs) in sublethally irradiated lymphopenic mice. These radio-resistant Tregs inhibited the induction of T_Es in tumor-draining lymph-nodes (TDLNs) during recovery from lymphopenia. The transfer of T_Ns into lymphopenic tumor-bearing mice resulted in some antitumor effects; however, Treg depletion after whole-body irradiation and reconstitution strongly inhibited tumor progression. Further analyses revealed that tumor-specific T cells were primed from the transferred T_Ns, whereas the Tregs originated from irradiated recipient cells. As in irradiated lymphopenic mice, a high percentage of Tregs was observed in cyclophosphamide-treated lymphopenic mice. The inhibition of Tregs in cyclophosphamide-treated mice significantly reduced tumor growth. These results indicate that the Tregs that survive cytotoxic therapies suppress antitumor immunity during recovery from lymphopenia and suggest that approaches to deplete radio and chemo-resistant Tregs can enhance cancer immunotherapies.