Temozolomide (TMZ) is commonly used in glioblastoma (GBM) chemotherapy. However, a great challenge for TMZ treatment is the rapid development of resistance and subsequent tumor recurrence and poor outcome. In the present study we established TMZ-resistant GBM cells (U87-TR and U251-TR) and found that the expression of PomGnT1 was significantly upregulated in TMZ-resistant GBM cells compared with the TMZ-sensitive counterparts. Furthermore, overexpression of PomGnT1 in U87-MG and U251-MG cells led to increased IC50 values for TMZ and reduced apoptosis of cells. Knockdown of PomGnT1 in both U87-TR and U251-TR cells led to decreased IC50 values for TMZ and enhanced apoptosis. Biochemical analysis revealed that PomGnT1 regulates the expression of factors in epithelial-mesenchymal transition signaling including TCF8, vimentin, β-catenin and Slug in GBM cells. These findings demonstrate that PomGnT1 might be a new focus of GBM research for treatment of recurrent TMZ-resistant GBM.