Abstract: Objective To observe effects of Biejiajian Pills on hepatocarcinoma (HCC) cell vasculogenic mimicry (VM) and explore the molecular mechanism by which Biejiajian Pills inhibits HCC metastasis and invasion. Methods Forty male SD rats were randomly divided into 4 groups for gastric lavage of normal saline or high, moderate or low doses of Biejiajian Pills (twice daily) for 4 consecutive days. The sera were collected from the rats for treatment of cultured human HCC HepG2 cells. VM formation in the cells was detected using an image acquisition and analysis system 24 h after incubation of the cells with the sera and with the RhoA/ROCK inhibitor Y-27632(P). The expression levels of RhoA and ROCK1 in the cells were detected using Western blotting, and the contents of VE-cadherin and PI3K in the culture supernatant were determined using ELISA. Results Treatment with the sera from Biejiajian Pills-treated rats significantly inhibited formation of VM in HepG2 cells, and the diameters of VM formed were significantly greater than those in the positive control group (P<0.01). Y-27632 completely inhibited the formation of VM in HepG2 cells (P<0.01). Treatments with Biejiajian Pills and Y-27632 both inhibited the expression of RhoA and ROCK1 (P<0.05) and significantly lowered the contents of VE-cadherin and PI3K in the culture supernatant (P<0.05). Conclusion Biejiajian Pills can inhibit the formation of VM in HCC cells in vitro possibly by inhibiting the RhoA/ROCK pathways and the expressions of VE-cadherin and PI3K.