Sinomenine (SIN) is an extract obtained from the plant Sinomenium acutum, which has been reported in hepatocellular carcinoma (HCC) and many other different types of human cancers as an anti-tumour agent. However, the molecular mechanism modulated or altered by SIN in HCC progression remains to be investigated. This study aims to uncover the molecular mechanism contributing to the tumour-suppressing effect of SIN in HCC. At first, HCC cells were treated with SIN for functional detections. Results of functional assays demonstrated that SIN was an efficient inhibitor of HCC cell viability, migration, and invasion. Next, O-GlcNAcylation and its removal OGA were found to be regulated by SIN treatment in HCC cells. According to rescue functional assays, OGA knockdown strengthened HCC cell viability, migration, and invasion suppressed by SIN. Moreover, as detected by mechanism experiments, OGA directly interacted with SP1 and blocked SP1 O-GlcNAcylation at site T407, indicating that SP1 was downregulated by OGA-mediated deglycosylation. Additionally, silencing of SP1 recovered the suppressing effects of SIN on HCC cell proliferation, migration, and invasion attenuated by OGA knockdown. Finally, SIN treatment inhibited the tumour tissue growth in vivo. In summary, this study unmasked the anti-tumour effect of SIN in HCC and SIN exerted functions by regulating OGA-mediated downregulation of SP1.