A number of human xenograft orthotopic models of hepatocellular carcinoma (HCC) have been previously established by growing histologically-intact patient specimens in nude mice. However, the availability of HBV and HCV negative human hepatocellular carcinoma specimens is scarce and the pattern of tumor growth in nude mice varies depending on the tumor type. In the present study, we have established a reproducible xenograft orthotopic model using a human HCC cell line designated HuT7-3 that was derived from two rounds of subcloning of the parental Huh-7 cell line. The tumor growth rate of the HuT7-3 cell line, grown at a primary subcutaneous site, was markedly higher than that of the Huh-7 parental cell line or the human hepatoblastoma Hep-G2 cell line. Furthermore, we have shown that doxorubicin, when administered intravenously, is efficient in inhibiting the development of subcutaneous tumor but leads to the regression of the orthotopic human HCC. Consequently, this novel HCC xenograft orthotopic model can be used for the evaluation of antitumor drugs.