We have previously shown in a model of claudin-low breast cancer that regulatory T cells (T regs) are increased in the tumor microenvironment (TME) and express high levels of PD-1. In mouse models and patients with triple-negative breast cancer, it is postulated that one cause for the lack of activity of anti–PD-1 therapy is the activation of PD-1–expressing T regs in the TME. We hypothesized that the expression of PD-1 on T regs would lead to enhanced suppressive function of T regs and worsen antitumor immunity during PD-1 blockade. To evaluate this, we isolated T regs from claudin-low tumors and functionally evaluated them ex vivo. We compared transcriptional profiles of T regs isolated from tumor-bearing mice with or without anti–PD-1 therapy using RNA sequencing. We found several genes associated with survival and proliferation pathways; for example, Jun, Fos, and Bcl2 were significantly upregulated in T regs exposed to anti–PD-1 treatment. Based on these data, we hypothesized that anti–PD-1 treatment on T regs results in a prosurvival phenotype. Indeed, T regs exposed to PD-1 blockade had significantly higher levels of Bcl-2 expression, and this led to increased protection from glucocorticoid-induced apoptosis. In addition, we found in vitro and in vivo that T regs in the presence of anti–PD-1 proliferated more than control T regs. PD-1 blockade significantly increased the suppressive activity of T regs at biologically relevant T reg/T naive cell ratios. Altogether, we show that this immunotherapy blockade increases proliferation, protection from apoptosis, and suppressive capabilities of T regs, thus leading to enhanced immunosuppression in the TME.