Previous studies have shown the potent anti-tumor effect of paeonol, a natural compound extracted from traditional Chinese herb medicine. However, the therapeutic effect and underlying mechanisms of paeonol in melanoma are largely unknown. Two melanoma cell lines (M14 and A375) were cultured in the presence of paeonol. Cell proliferation was determined by colony formation and cell-counting kit 8 assays. Real-time quantitative reverse transcription polymerase chain reaction and Western blot were performed to detect mRNA and protein expression, respectively. Paeonol treatment significantly inhibited the colony formation and proliferation of melanoma cells by enhancing the expression of the well-characterized tumor suppressor microRNA (miR)-143. Block of miR-143 diminished the inhibitory effects of paeonol on cell growth. miR-143 directly bound to Wnt family member 5B (WNT5B) gene. Paeonol therefore down-regulated WNT5B expression through miR-143. Moreover, paeonol suppressed melanoma growth in vivo. Our study reveals that paeonol effectively suppresses melanoma cell growth both in vitro and in vivo through miR-143/WNT5B axis, which shows potential for the treatment of melanoma.