Updated project metadata. O-GlcNAc glycosylation is a prevalent protein post-translational modification (PTM) that occurs intracellularly. Previous investigations have demonstrated that O-GlcNAcylation crosstalks with phosphorylation and ubiquitination, but it is unclear whether it interplays with other PTMs. Here we studied its relationship with ADP-ribosylation, which involves decorating target proteins with the ADP-ribose moiety. We discovered that one ADP-ribosylation “eraser”- ADP-ribose glycohydrolase (PARG)- is O-GlcNAcylated at Ser26. O-GlcNAcylation of PARG is essential to maintain its nuclear localization and chromatin association. In hepatocellular carcinoma (HCC) cells, PARG O-GlcNAcylation enhances DNA damage-binding protein 1 (DDB1) poly(ADP-ribosyl)ation (PARylation) and attenuates its ubiquitination. DDB1 is thus stabilized and degrades its downstream targets, such as c-Myc. We further utilized mouse xenograft models and demonstrated that PARG-S26A promoted HCC. Our study thus revealed that PARG O-GlcNAcylation inhibits HCC, and we propose that O-GlcNAc glycosylation may crosstalk with many other PTMs.