Portal vein invasion (PVI) is common in hepatocellular carcinoma (HCC) and largely contributes to tumor recurrence after radical tumor resection or liver transplantation. Vasculogenic mimicry (VM) was an independent vascular system lined with tumor cells and associated with poor prognosis of HCC. The present study was conducted to evaluate the relationship between VM and portal vein invasion. A total of 44 HCC cases receiving anatomic liver resection were included in the study and were divided into groups with and without PVI. The prevalence of VM in each group was examined by CD34-PAS dual staining. The regulatory molecules of VM formation such as Notch1, Vimentin and matrix metalloproteinases (MMPs) were investigated by immunohistochemical staining. Analysis was performed to explore the association of PVI, VM and the VM regulatory molecules. PVI was found in 40.91% (18/44) cases and VM was found in 38.64% (17/44) cases in total samples. The incidence of VM was 72.22% (13/18) in PVI group while it was 15.38% (4/26) in non-PVI group (P<0.001), VM formation was positively correlated with PVI (r=0.574, P<0.001). The VM forming regulatory molecules such as Notch1, Vimentin, MMP-2 and MMP-9 were found to be correlated with PVI in HCC patients. Taken together, our results suggested that VM formation, alone with its regulatory molecules, is the promoting factor of PVI in hepatocellular carcinoma.