ABSTRACT: Cervical cancer (CC) is a common and deadly malignancy among women worldwide, characterized by high mortality and poor prognosis. Glycosyltransferases (GTs) are enzymes that catalyze glycosyl transfer reactions and play significant roles in cancer development and progression. However, their role in cervical cancer remains unclear. METHODS: This study combined single-cell RNA sequencing (scRNA-seq) data and spatial transcriptomics (stRNA-seq) to explore the role of GTs in cervical cancer. We utilized comprehensive scoring algorithms to evaluate GT expression, conducted cell communication analysis, and identified differentially expressed GT-related genes in tumor-associated epithelial cells. Functional assays were performed to assess the impact of GALNT3 expression on cervical cancer cell proliferation and invasiveness. RESULTS: Our analysis revealed that GT-related genes are highly enriched in cervical cancer epithelial cells. We identified 11 differentially expressed GT-related genes, including GALNT3, which showed distinct characteristics in epithelial cells. GALNT3-negative epithelial cells were found to be a protective factor for cervical cancer patients, showing a significant negative correlation with tumor-associated macrophages and myeloid-derived suppressor cells. Knockdown or overexpression of GALNT3 significantly affected cell proliferation and invasiveness in C-33A and Caski cell lines. CONCLUSION: This study highlights the critical role of GTs, particularly GALNT3, in the progression and prognosis of cervical cancer. GALNT3-negative epithelial cell infiltration could be a favorable factor for cervical cancer prognosis, providing new insights for therapeutic strategies targeting GTs in cervical cancer.