Cells in the tumor microenvironment (TME) communicate via membrane-bound and secreted proteins, which are mostly glycosylated. Altered glycomes of malignant tumors influence behaviors of stromal cells. In this study, we showed that the loss of core-1 β1,3-galactosyltransferase (C1GALT1)-mediated O-glycosylation suppressed tumor growth in syngeneic head and neck cancer mouse models. O-glycan truncation in tumor cells promoted the M1 polarization of macrophages, enhanced T-cell-mediated cytotoxicity, and reduced interleukin-6 (IL-6) levels in the secretome. Proteasomal degradation of IL-6 was controlled by the O-glycan at threonine 166. Both IL-6/IL-6R blockade and O-glycan truncation in tumor cells induced similar pro-inflammatory phenotypes in macrophages and cytotoxic T lymphocytes (CTLs). The combination of the O-glycosylation inhibitor itraconazole and anti-programmed cell death protein 1 (anti-PD-1) antibody effectively suppressed tumor growth in vivo. Collectively, our findings demonstrate that O-glycosylation in tumor cells governs their crosstalk with macrophages and CTLs. Thus, targeting O-glycosylation successfully reshapes the TME and consequently enhances the efficacy of anti-PD-1 therapy.