BACKGROUND: Our previous studies revealed a critical role of a novel CTLA4-protein kinase C-eta (PKCeta) signaling axis in mediating the suppressive activity of regulatory T cells (Tregs) in antitumor immunity. These studies have employed adoptive transfer of germline PKCeta-deficient (Prkch (-/-)) Tregs into Prkch (+/+) mice prior to tumor implantation. Here, we extended these findings into a biologically and clinically more relevant context. METHODS: We have analyzed the role of PKCeta in antitumor immunity and the tumor microenvironment (TME) in intact tumor-bearing mice with Treg-specific or CD8(+) T cell-specific Prkch deletion, including in a therapeutic model of combinatorial treatment. In addition to measuring tumor growth, we analyzed the phenotype and functional attributes of tumor-infiltrating immune cells, particularly Tregs and dendritic cells (DCs). RESULTS: Using two models of mouse transplantable cancer and a genetically engineered autochthonous hepatocellular carcinoma (HCC) model, we found, first, that mice with Treg-specific Prkch deletion displayed a significantly reduced growth of B16-F10 melanoma and TRAMP-C1 adenocarcinoma tumors. Tumor growth reduction was associated with a less immunosuppressive TME, indicated by increased numbers and function of tumor-infiltrating CD8(+) effector T cells and elevated expression of the costimulatory ligand CD86 on intratumoral DCs. In contrast, CD8(+) T cell-specific Prkch deletion had no effect on tumor growth or the abundance and functionality of CD8(+) effector T cells, consistent with findings that Prkch (-/-) CD8(+) T cells proliferated normally in response to in vitro polyclonal or specific antigen stimulation. Similar beneficial antitumor effects were found in mice with germline or Treg-specific Prkch deletion that were induced to develop an autochthonous HCC. Lastly, using a therapeutic model, we found that monotherapies consisting of Treg-specific Prkch deletion or vaccination with irradiated Fms-like tyrosine kinase 3 ligand (Flt3L)-expressing B16-F10 tumor cells post-tumor implantation significantly delayed tumor growth. This effect was more pronounced in mice receiving a combination of the two immunotherapies. CONCLUSION: These findings demonstrate the potential utility of PKCeta inhibition as a viable clinical approach to treat patients with cancer, especially when combined with adjuvant therapies.