Transendothelial migration is a pivotal step before the dissemination of tumor cells into the blood circulation. Related researches about the crosstalk between tumor cells and endothelial cells could contribute to understanding the mechanism of transendothelial migration. Cumulative studies showed that CD133 was an important marker for cancer stem cells. In our research, a co-culture system was developed to study the interaction between CD133+ liver cancer cells and human umbilical vein endothelial cells. The results showed that the direct co-cultured supernatants promoted the migration and invasion of CD133+ liver cancer cells. It was further investigated that the expression level of chemokine CXCL9 was significantly elevated in the culture supernatants of direct co-culture system by activating the NF-kB, rather than in the indirect co-culture system or mono-culture system. High expression of CXCL9 in the direct co-cultured supernatants played a significant role in enhancing the migration and invasion of CD133+ liver cancer cells. Collectively, these findings suggest that chemokine CXCL9 may function as a potential target during the process of transendothelial migration.