Objective To investigate the effect of NOTCH4 expression on vasculogenic mimicry( VM) formation in hepatocellular carcinoma( HCC). Methods Tumor tissues were collected from 85 patients diagnosed with HCC. The relationship between NOTCH4 expression and VM was examined using immunohistochemical staining and PAS / CD31 double staining. The influence of siRNA- mediated NOTCH4 silencing on VM network formation in HCC was observed in the 3D cell culture system in vitro. Experimental data were assessed with Mann-Whitney U test and one- way analysis of variance. Results NOTCH4 had significantly higher expression in VM- positive HCC tissues than in VM- negative HCC tissues( P = 0. 019). In siRNA- treated cells,NOTCH4 mRNA and protein expression was significantly down- regulated compared with those in mock- treated cells( P = 0. 007; P = 0. 003) and untreated cells( P = 0. 000; P = 0. 000). VM network formation was impaired by inhibition of NOTCH4 expression in the 3D cell culture system. Conclusion NOTCH4 plays an important role in VM formation in HCC and may provide a novel molecular target for anti- angiogenesis therapy for HCC.