CD4⁺FoxP3⁺ regulatory T cells (Tregs) have been shown to suppress T cell–mediated host immune responses against self- and nonself-antigens; however, the impact of CD4⁺ Tregs on human antitumor immune responses and their influence on cancer treatment are unknown. In the present study, we explored the factors that influence CD4⁺ Treg reconstitution in patients receiving adoptive immunotherapy following conditioning regimens designed to enhance T-cell function and evaluated potential associations between CD4⁺ Treg levels and clinical responses to therapy. The analysis of 4 trials employing nonmyeloablative chemotherapy with or without total body irradiation (TBI) before adoptive T-cell transfer revealed that the percentage and number of reconstituting CD4⁺FoxP3⁺ Tregs observed in the peripheral blood was higher in nonresponders than in responders. The addition of TBI resulted in a further depletion of CD4⁺ Tregs, and the degree of depletion was dependent on the TBI dose. The number of administered doses of IL-2 was found to be positively associated with peripheral Treg reconstitution. These observations provide strong evidence that endogenous CD4⁺ Tregs have a negative impact on cancer therapy, and suggest that strategies reducing Treg levels may provide clinical benefit to cancer patients. All 5 clinical trials are registered at www.clinicaltrials.gov as NCT00001832, NCT00096382, NCT00335127, NCT00509496, and NCT00513604.