Human ribonuclease inhibitor (RI) is an acidic cytoplasmic glycoprotein with molecular mass of 50 ku. RI can inhibit the activity of ribonuclease A (RNase A). Angiogenin (Ang) is a member of RNase A superfamily. RI also can inhibit Ang activities by tight combination. Angiogenesis is an essential condition for the development of tumors and their metastatic dissemination. So anti-angiogenesis will be an efficient method in the inhibition of the growth and metastasis of tumor. The experiment demonstrated that RI might effectively block the angiogenesis that was induced by angiogenin. RI is constructed almost entirely of leucine-rich repeats that might involve in other unclear biological effect. In order to understand further the potential function of RI and investigate the role of RI in invasion and metastasis. The study established a transfection of human RI cDNA into B16 melanoma cells by the retroviral packaging cell line PA317 carrying the pLNCX-RI in vitro. Transfected B16 cells by PA317 carrying the pLNCX and untransfected B16 cells were used as control. The B16pLNCX-RI cell line with a stably high expression of RI was identified by PCR, RT-PCR, Western blot and immunofluorescence assay respectively. The results showed that the transfected RI gene might significantly inhibit cell proliferation, migration, and enhance cell adhesion, as well as, make morphological changes in vitro. Cell doubling time were (24.98±0.16)h, (25.62±0.28)h, (32.64±1.11)h in B16 cells, B16 pLNCX and B16 pLNCX-RI cells respectively. Cell adhesion rate was significantly increased by 19.5% and 17.8% as well as cell migration was reduced by 60% and 61.4% in B16 pLNCX-RI cells compared with pLNCX B16 cells and B16 cells respectively. B16 pLNCX-RI cells became flatter, less nucleoli, less division phases and weaker alkalophilic quality of cytoplasm compared with control groups, which should imply that cell proliferation viability was decreased and malignant phenotype was improved on the cell transfected RI. Mice injected with B16 pLNCX-RI cells show a significant inhibition of the metastasis of tumor with lighter lung weight, fewer metastasis nodules, a lower incidence rate, a lower density of blood vessels and longer survival with respect to the control groups, which implied that RI might be involved in metastasis of melanoma. The results of experiments show RI has a significant antitumor metastasis effect and suggest that it is partially responsible for inhibiting angiogenesis, decreasing cell proliferation, reducing cell migration and enhancing cell adhesion.