In our previous study, we reported that the cannabinoid receptors CB1 and CB2 are overexpressed in human hepatocellular carcinoma (HCC) tissues. Recently, the antitumor potential of the endogenous cannabinoid anandamide (AEA) has also been addressed. The present study was conducted to investigate the anti-proliferative effects of AEA in HCC cells. The human HCC cell line Huh7 was used. Cell proliferation was measured by MTT assay and flow cytometry. Apoptotic analysis was investigated by TUNEL assay. Real-time PCR and western blot analysis were used to analyze the expression of relevant molecules. The results of this study demonstrated that AEA inhibited the proliferation of Huh7 cells, resulted in G1 cell cycle arrest and induced apoptosis. Furthermore, downregulation of CDK4 and upregulation of p21 and Bak by AEA were observed. This study defines the anti-proliferative effects of anandamide in HCC cells and suggests that AEA has therapeutic potential in the management of HCC patients.