PPP2R2A is one of the regulatory subunits of the PP2A phosphatase complexes, and previous studies showed that its upregulation promotes cancer cell survival and growth. In this research, we used the tandem affinity purification and the HPLC-Chip-ESI/MS/MS mass spectrometry to screen the PPP2R2A-binding proteins and the results indicated that the GFPT-1/-2 were the potential partners of PPP2R2A. We further validated the interaction between PPP2R2A and GFPT-1/-2 through GST Pull-down, co-immunoprecipitation and immunofluorescence assays. And we found that knockdown of PPP2R2A by lentivirus-mediated shRNA enhanced the phosphorylation of GFPT2, whereas the phosphorylation of GFPT1 had no significant change. GFPT2 is a rate-limiting enzyme in the hexosamine pathway. Our results showed that the knockdown of PPP2R2A promoted the total cellular O-GlcNAcylation in MDA-MB-231 breast cancer cells. These results suggest that PPP2R2A interacts with GFPT1/2, and leads to the phosphorylation of GFPT2, which can regulate the cellular O-GlcNAcylation.