Objective To observe the suppressive effect of 3,5-dichlorophenylbiguanide on pulmonary metastasis of melanoma in vivo. MethodsHuman melanoma cell line M21 cells were injected into the tail vein of nude mice (defined as day 1). The treatment procedure was carried out at days 5,9, 3,7, and 21 by tail vein injection of 3,5-dichlorophenylbiguanide at a dose of 6 mg/kg, 12 mg/kg or 24mg/kg to the nude mice of the experimental groups. In addition, positive control, negative control, and vehicle control groups were set up. The mice were sacrificed at day 50. The lungs were removed and fixed in Bouin’s fixative solution for 24 h. Metastasis scores were figured out by a formula according to the diameters and numbers of white metastatic nodules on the lung surface. ResultsThe 3,5-dichlorophenylbiguanide treatment reduced the metastasis scores to 55.25±13.60 (6 mg/kg), 35.13±17.36 (12 mg/kg) and 12.83±11.44 (24 mg/kg), significantly lower than that of the vehicle control group (P<0.05). The cRGDfV treatment reduced metastasis score to 11.50±10.44, significantly lower than that of the vehicle control group (P<0.05). The metastasis score of the negative control IH1066 treatment group was 88.50±17.21, and that of the untreated group was 88.88±11.29, both with a non-significant difference compared with that of the vehicle control group (82.50±17.72) (P>0.05). Conclusion3,5-dichlorophenylbiguanide shows a suppressive effect on the pulmonary metastasis of melanoma in mice in a dose-dependent manner.