Abstract: Objective To invest the inhibitory effect of tumor growth and the expression of vascular endothelial growth factor (VEGF) by acetoacetate extractive of Celastrus orbiculatus Thunb in orthotropic human liver carcinoma nude mice model. Methods Human HCC HepG2 cells transduced with red fluorescent protein (RFP-HepG2) were inoculated into the liver of BALB/c nude mice. The tumor-bearing nude mice were randomly divided into five groups: control group (G1 group), Oxaliplatin positive control group (G2 group, 25 mg/kg), COT low dose group (G3 group, 20 mg/kg), COT high dose group (G4 group, 40 mg/kg) and COT early treatment group (G5 group, 20 mg/kg). G5 group of mice was treated with COT p.o. from day 2 post tumor implantation. All other mice were treated from day 20 post tumor implantation. The growth of xenografted tumors was traced weekly by in vivo fluorescence imaging technology. Real time whole body fluorescence imaging was performed to measure tumor growth during the study. At the end of treatment, all of mice were killed and tumor tissue were collected. VEGF expression in the tumors collected at autopsy was analyzed by immunohistochemical staining. VEGF mRNA expression in the tumors collected at autopsy was analyzed by reverse transcription-polymerase chain reaction (RT-PCR). Results The COT significantly inhibited the growth of xenografted tumors. The inhibitory effect of COT in G5 group was superior to those in G3 group and G4 group, which was almost close to G2 group. VEGF protein was found to be expressed in the cytoplasm of all liver cells and the cell membrane of some liver cells. Average optical density (D) of the tumor VEGF expression was significantly reduced by high-dose COT and early COT treatment, compared to G1 group and G3 group (t = 4.657, 6.440, 7.712, 8.900; P = 0.0037, 0.0031, ＜ 0.001, ＜ 0.001). The tumors from early treatment with COT and oxaliplatin-treated mice had significantly lower VEGF expression levels compared to all other COT-treated mice. Consistent with VEGF protein expression by immunohistochemistry, the tumor VEGF mRNA expression was significantly reduced by G4 group and G5 group, compared to G1 group and G3 group (t = 6.320, 7.563, 12.907, 12.545; P = 0.0002, 0.0003, ＜ 0.001, ＜ 0.001). Conclusions The acetoacetate extractive of C. articulatus Thunb possess the effect of anti-tumor in vivo. The COT inhibits the growth of tumor. Inhibit VEGF expression by COT may inhibit angionesis and then may contribute in part to the inhibition of tumor growth.