Invariant natural killer T (NKT) cells produce large amounts of cytokines, such as IFN-γ, while simultaneously exerting cytotoxic activity upon activation of their invariant TCR by recognizing α-galactosylceramide (α-GalCer) presented on CD1d. NKT cell-based immunotherapy, which involves adoptive transfer of α-GalCer-pulsed dendritic cells (DCs) to activate iNKT cells, has been developed for treating cancer patients, as NKT cells play an essential role in antitumor immunity. The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that regulates various immune responses and is often associated with immunosuppression. As AhR ligands are abundant in the tumor microenvironment, we hypothesized that blocking AhR signaling with the AhR antagonist CH223191 might enhance the antitumor efficacy of NKT cell-based immunotherapy. Intraperitoneal administration of CH223191 enhanced the efficacy of NKT cell-based immunotherapy and suppressed tumor growth in a CT26 colon tumor mouse model in vivo. Notably, depletion of CD8+ T cells abrogated the CH223191-mediated enhancement of NKT cell antitumor activity. Furthermore, CH223191 treatment increased infiltration of CD45+ cells into necrotic tumor areas. These findings suggest that AhR signaling could represent a novel therapeutic target in NKT cell-based immunotherapy.