CCN family, a group of six extracellular matrix-associated proteins, plays an important role in fibrosis. WISP3 has addressed as a pro-fibrotic molecule in the development of human lung fibrosis. However, whether WISP3 involved in the activation and proliferation of renal fibroblast, and ultimately inhibited fibroblast–myofibroblast transdifferentiation remained unknown. Herein, we found that down-regulated WISP3 was involved in the fibrogenesis of rat renal NRK-49F cells induced by transforming growth (TGF-β1), which was further confirmed in a rat renal fibrosis induced by unilateral ureteral obstruction (UUO). In the present study, we aimed to investigate the roles of WISP3 in NRK-49F fibroblast–myofibroblast transdifferentiation, and the underlying mechanism. Results showed that after TGF-β1 treatment, significant increased cell proliferation, and up-regulated expressions of TGF-β1, connective tissue growth factor (CTGF), α-smooth muscle actin (α-SMA), vimentin, as well as increased concentrations of collagen types I (COL I), collagen types III (COL III) and hydroxyproline in cell culture supernatant were observed, demonstrating a successful establishment of fibroblast–myofibroblast transdifferentiation of NRK-49F cells. Besides, siRNA-WISP3 remarkably promoted the fibrogenesis of NRK-49F cells with or without TGF-β1 treatment, and increased mRNA levels of Axin, demonstrating that activating WNT signaling pathway was the underlying mechanism. However, lentivirus-mediated WISP3 overexpression exerted an opposite effect, protecting NRK-49F cells from transdifferentiation, and decreasing mRNA levels of Axin. In conclusion, the WISP3 played an anti-fibrotic role in NRK-49F cells, and WNT signaling pathway was the potential mechanism. WISP3 was an anti-fibrotic factor in fibroblast–myofibroblast transdifferentiation, and may be used as a possible target for prevention and treatment of human renal fibrosis.