Regulatory T (Treg) cells have an immunosuppressive function and highly express the immune checkpoint receptor PD-1 in the tumor microenvironment; however, the function of PD-1 in tumor-infiltrating (TI) Treg cells remains controversial. Here, we showed that conditional deletion of PD-1 in Treg cells delayed tumor progression. In Pdcd1fl/flFoxp3eGFP−Cre-ERT2(+/−) mice, in which both PD-1-expressing and PD-1-deficient Treg cells coexisted in the same tissue environment, conditional deletion of PD-1 in Treg cells resulted in impairment of the proliferative and suppressive capacity of TI Treg cells. PD-1 antibody therapy reduced the TI Treg cell numbers, but did not directly restore the cytokine production of TI CD8+ T cells in TC-1 lung cancer. Single-cell analysis indicated that PD-1 signaling promoted lipid metabolism, proliferation and suppressive pathways in TI Treg cells. These results suggest that PD-1 ablation or inhibition can enhance antitumor immunity by weakening Treg cell lineage stability and metabolic fitness in the tumor microenvironment.