B3GNT5, a critical member of the β-1,3-N-acetylglucosaminyl transferase gene family involved in lactose and glycosphingolipids biosynthesis, has been documented to promote tumor-infiltrating T-cell responses. Our research utilized the Pan-Cancer dataset from The Cancer Genome Atlas (TCGA) to explore the functional role of B3GNT5. Our study demonstrated that the antibody-driven inhibition of B3GNT5 diminished T cell-mediated anti-tumor responses in both in vitro and in vivo settings. By analyzing RNA-seq data from Genotype-Tissue Expression (GTEx) and TCGA databases, we observed differential expression levels of B3GNT5 across various tumor types accompanied by an unfavorable prognostic correlation. We further utilized integrated clinical survival data from TCGA and immune cell infiltration scoring patterns to identify significant associations between B3GNT5 expression and immune checkpoints, cancer stemness, chemokines, chemokine receptors, and immune-activating genes. B3GNT5's expression was highly correlated with different immunoregulatory factors, including T cell infiltration, chemokine receptors, and activation genes. Subsequent experiments discovered that suppressing B3GNT5 expression in pancreatic adenocarcinoma cells significantly reduced their tumorigenicity by limiting sphere-forming ability and self-renewal capacity, thus underscoring B3GNT5's vital role as a prognostic factor in immune regulation across pan-cancer. Our findings suggest that B3GNT5 presents a viable target for cancer immunotherapy by enabling effective communication between cancer stem cells and immune cells during tumor treatment.