Objective: To investigate the effect and mechanism of tetrahydrobiopterin (BH4) on the angiogenesis in hepatocellular carcinoma (HCC). Methods: BALB/c-nu mice were subcutaneously injected with HepG-2 cells and randomly divided into control and BH4 groups. The BH4 group and control group received 20 mg/kg BH4 or saline by intraperitoneal injection daily for two weeks, respectively. The level of BH4 was measured by high performance liquid chromatography (HPLC), the level of nitric oxide (NO) was measured by Griess test array, the transcriptional level of K-ras was measured by quantitative RT-PCR, and the protein expressions of guanosine triphosphate cyclohydrolase Ⅰ(GTPCH), endothelial nitric oxide synthase (eNOS), phospho-Akt and Akt were determined by Western blot. Results: BH4 level in the tumor tissues of BH4 group was (0.24±0.02) μg/ml, significantly higher than the (0.17±0.01) μg/ml in the control group (P<0.01). The level of NO in the tumor tissues of BH4 group was (51.44±2.90) mmol/L, significantly higher than the (24.77±0.54) mmol/L in the control group (P<0.01). The tumor volume of BH4 group was (191.05±8.70) mm3, significantly higher than the (103.10±5.03) mm3 in the control group (P<0.01). The expressions of CD34, K-ras, phospho-eNOS, phospho-Akt and GTPCH were significantly up-regulated in the tumor tissues of BH4 group when compared with those of the control group (P<0.01). Conclusions: BH4 recognized as an essential cofactor of eNOS can increase tumor-produced NO by activating the wild-type Ras-PI3K/Akt pathway, thus induces angiogenesis. This might provide a novel and promising way to control the progression of hepatocellular carcinoma through targeting BH4 synthesis pathway and inhibiting angiogenesis.