Importantly, for the first time, we demonstrated that ppGalNAc-T4 modulated TGF-β signaling by directly catalyzing O-GalNAcylation of TGF-β type II receptor at Ser31 and then attenuated dimerization of TGF-β receptors, resulting in the inhibition of TGF-β signaling and EMT in breast cancer cells. This evidence concerns the gene TGFB1 and breast cancer.