Abstract In this study, we investigated the role of the Wnt/β-catenin signaling pathway in antitumor immune responses. We report that the concentration of secreted Wnt3a was significantly higher in conditioned medium from tumor or nontumor tissues obtained from all hepatocellular carcinoma or colorectal cancer patients tested, than in serum of healthy donors or patients. In addition, both Wnt3a and β-catenin were overexpressed by tumor-infiltrating and nontumor-infiltrating CD4+ or CD8+ T cells. The majority of these T cells expressed a dysfunctional effector memory Eomes+T-bet−phenotype that we defined as partially exhausted, because they performed effector functions (in terms of interferon-γ and tumor necrosis factor-α production, as well as CD107a mobilization) despite their PD-1 expression. Wnt3a/β-catenin signaling in T naïve cells in vitro recapitulated the T-cell setting in vivo. Indeed, the differentiation of cultured T naïve cells was arrested, producing cells that resembled the EomeshighT-betlowβ-cateninhigh T cells with moderate effector functions that infiltrated tumor and nontumor areas. Wnt3a blockade improved the capacity of T naïve cells to differentiate into effector cells in vitro. However, Wnt3a blockade did not affect the function and phenotype of differentiated, partially exhausted, tumor-infiltrating T cells ex vivo. Taken together, our data suggest that Wnt3a blockade halts the capacity of Wnt/β-catenin signaling to inhibit the differentiation of T naïve cells, but it does not restore the dysfunction of differentiated T cells, in the tumor setting. Cancer Immunol Res; 6(8); 941–52. ©2018 AACR.