Protein glycosylation, attaching glycans covalently onto amino acid side chains of protein by various glycosyltransferase, is the most common post-translational modification. The UDP-GalNAc transferase 3 (GANLT3), encoded by Galnt3, transfers N-acetyl-d-galactosamine to hydroxyl groups of the side chains of Ser/Thr residues, initiating mucin type O-glycosylation of proteins. Most researches as yet focus on the involvement and abnormal expression of GALNT3 in various tumors. In this study, we found that GALNT3 was significantly decreased in the lungs after influenza A virus (IAV) infection in mice. Overexpression of GALNT3 in cell lines markedly inhibited IAV replication. Further experiments demonstrated that GALNT3 inhibited NF-κB signaling by preventing the translocation of phosphorylated P65 into nucleus. Therefore, our results reveal an important role of GALNT3 in regulating host responses during IAV infection, indicating the broad functions of the GALNT family, and the direct involvement of GALNTs during viral infections.