Pancreatic ductal adenocarcinoma (PDAC) is characterized by poor prognosis and high mortality. Transforming growth factor-β (TGF-β) plays a key role in PDAC tumor progression, which is often associated with aberrant glycosylation. However, how PDAC cells respond to TGF-β and the role of glycosylation therein is not well known. Here, we investigated the TGF-β-mediated response and glycosylation changes in the PaTu-8955S (PaTu-S) cell line deficient in SMA-related and MAD-related protein 4 (SMAD4), a signal transducer of the TGF-β signaling. PaTu-S cells responded to TGF-β by upregulating SMAD2 phosphorylation and target gene expression. We found that TGF-β induced expression of the mesenchymal marker N-cadherin but did not significantly affect epithelial marker E-cadherin expression. We also examined differences in N-glycans, O-glycans, and glycosphingolipid-linked glycans in PaTu-S cells upon TGF-β stimulation. TGF-β treatment primarily induced N-glycome aberrations involving elevated levels of branching, core fucosylation, and sialylation in PaTu-S cells, in agreement with TGF-β-induced changes in the expression of glycosylation-associated genes. In addition, we observed differences in O glycosylation and glycosphingolipid glycosylation profiles after TGF-β treatment, including lower levels of sialylated Tn antigen and neoexpression of globosides. Furthermore, the expression of transcription factor sex-determining region Y-related high-mobility group box 4 was upregulated upon TGF-β stimulation, and its depletion blocked TGF-β-induced N-glycomic changes. Thus, TGF-β-induced N-glycosylation changes can occur in a sex-determining region Y-related high-mobility group box 4-dependent and SMAD4-independent manner in the pancreatic PaTu-S cancer cell line. Our results open up avenues to study the relevance of glycosylation in TGF-β signaling in SMAD4-inactivated PDAC.