O-linked-ß-N-acetylglucosamine (O-GlcNAc) modification is a crucial post-translational modification. The enzymes responsible for the addition and removal of O-GlcNAc have been identified as O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). In this study, O-GlcNAcylation level was examined in forty hepatocellular carcinoma (HCC) tissues of patients who underwent liver transplantation (LT) and ten healthy liver tissues by immunohistochemistry analysis. We also examined the expression of OGT and OGA in sixty HCC samples using real-time reverse-transcription polymerase chain reaction and analyzed their correlations with clinical parameters and prognosis in sixty HCC patients treated with LT. Additionally, the global O-GlcNAcylation level was altered through OGT and OGA silencing in the HCC cell line, and the effects of O-GlcNAcylation on cancer malignancy were investigated. We found that the global O-GlcNAcylation levels were significantly elevated in HCC tissues than that in healthy liver tissues (P = 0.031); moreover, O-GlcNAcylation was significantly enhanced in the tumor tissues of patients who had suffered from HCC recurrence after LT compared with those who had not (P = 0.046). Importantly, low expression of OGA was an independent prognostic factor for predicting tumor recurrence of HCC following LT (P = 0.041, hazard ratio, 0.438), especially in AFP low patients. In vitro assays demonstrated that O-GlcNAcylation play important roles in migration, invasion, and viability of HCC cells, partly through regulating E-cadherin, MMP1, MMP2, and MMP3 expression. Altogether, these results suggest that O-GlcNAcylation might play important roles in HCC formation and progression and may be a potential marker to predict patient risk of recurrence after LT and a valuable target for therapy.