Immunotherapy is expected to become the most promising new treatment for ovarian cancer owing to its immunogenicity. However, immunosuppression in the tumor microenvironment is a major obstacle to the efficacy of tumor therapy. Studies have found different metabolism ways of regulatory T cells (Tregs) in the cancer environment may be related to the immunosuppression and Toll-like receptor 8 (TLR8) can reverse the suppression function of Tregs. But it is still unclear that if the TLR8-mediated function reversal is associated with the change of glucose metabolism of Tregs. It was found that the positive expression rates of Glut1, HIF-1α, and Ki67 in CD4+ Treg cells of OC were significantly higher than that in benign ovarian tumor and HC, and also significantly higher than that in CD4+ Teffs of OC. What's more, compared with CD4+ Teff group, CD4+ Tregs highly expressed seven genes and three proteins related to glucose metabolism and had higher levels of glucose uptake and glycolysis. After activating TLR8 signal of CD4+ Tregs, the proliferation level of naive CD4+ T cells was higher than that of the control group. At the same time, the expression levels of eight genes and five proteins related to glucose metabolism in CD4+ Treg cells with TLR8 activated were decreased and levels of glucose uptake and glycolysis were also lower. Furthermore, TLR8 signaling also downregulated the mTOR pathway in CD4+ Tregs. CD4+ Tregs pretreated with 2-deoxy-d-Glucose (2-DG) and galloflavin also attenuated the inhibition of Teffs proliferation. Although CD4+ Tregs pretreated with 2-DG and galloflavin before activating TLR8 signal had no significant difference compared with the group only treated with inhibitors, which suggested TLR8-mediated reversal of CD4+ Treg cells inhibitory function in ovarian cancer cells co-cultured microenvironment had a causal relationship with glucose metabolism.