Background: The immune responses play significant roles in the onset, progression, and outcome of oral squamous cell carcinoma (OSCC). CD4 + regulatory T cells (Tregs) significantly impact tumor immunity. However, their role in OSCC development remains elusive. Methods: In a carcinogen-induced mouse OSCC model, interleukin-23 receptor (IL-23R) expression on Tregs and Treg function were determined by flow cytometry. IL-23R overexpression in Tregs was achieved by lentiviral infection, followed by evaluation of the expression of Forkhead box P3 (Foxp3), T-bet, retineic-acid-receptor-related orphan nuclear receptor gamma t, and cytokines by flow cytometry. Adoptive transfer assays were applied to analyze the function of IL-23R − overexpressing Tregs in vivo. The cellular sources of IL-23 were also determined by flow cytometry. Results: IL-23R − Tregs and IL-23R + Tregs were found in the tongues but not spleens of OSCC-bearing mice. IL-23R + Tregs expressed lower Foxp3 but higher T-bet than IL-23R − Tregs. IL-23R − Tregs produced abundant IL-10 and transforming growth factor (TGF)-β, while IL-23R + Tregs produced lower IL-10 and TGF-β but remarkably higher interferon (IFN)-γ. Furthermore, IL-23R + Tregs possessed more phosphorylated signal transducer and activator of transcription (STAT3) and STAT4 than IL-23R − Tregs. IL-23R + Tregs were less immunosuppressive than IL-23R − Tregs, as evidenced by weaker inhibition of activated conventional T cells. IL-23R overexpression in splenic Tregs remarkably reduced the expression of IL-10 and TGF-β but increased IFN-γ expression when Tregs were adoptively transferred into OSCC-bearing mice. In the OSCC microenvironment, macrophages, dendritic cells, and malignant OSCC cells produced IL-23 which might modulate the function of IL-23R + Tregs. Conclusions: This study unveils Treg heterogeneity, thus deepening the understanding of Treg biology and tumor immunity in OSCC.