Background: Adipose-derived stem cells ( ADSCs ) therapy is considered as a promising alternative to treat osteoarthritis (OA).Considerable evidence has shown that the Wnt signaling pathway is involved in chondrogenic differentiation in various stem cells.In the present study, we explored in detail the mechanism of of Wnt signaling pathway in the regulation of adipose-derived stem cell chondrogenic differentiation.Methohs: Adipose-derived stem cells were extracted from Sprague-Dawley groin area, isolated, cultured and verified. Then LiCl and DKK-1 used to induce ADSCs cartilage differentiation to detect indicators of cartilage at different stages, such as collagen type II (Collagen2a), aggrecan (Aggrecan) and Wnt pathway key proteins Sox9, β-catenin, Glycogen synthase kinase-3β (GSK-3β) expression.Subsequently, lentivirus was used as the vector, the full-length Sox9 gene was transfected into ADSCs and the expression levels of relevant indicators were detected again after stable expression.Results: In the process of inducing differentiation, the Wnt pathway promoted the rapid proliferation of ADSCs in the early, middle and late stages, and up-regulates Sox9 expression.Cartilage indicators Collagen 2a and Aggrecan expression levels were not significantly increased in the early stage, but significantly increased in the middle and late stages (P <0.05). At the later stage, however, the Wnt pathway down-regulated Sox9 expression, weakened the mature cartilage phenotype, promoted cartilage hypertrophy and early cartilage ossification. Finally, the overexpression of Sox9 feedback inhibits the Wnt pathway activity, down-regulates β-catenin expression, maintains cartilage phenotype, delays cartilage hypertrophy and early osteogenesis.Conclusion:The Wnt pathway regulates chondrogenic differentiation of ADSCs by regulating Sox9, but its regulatory mechanism is significantly different at different stages of induced differentiation.