This study aimed to reveal the cross association of the Wnt-Smad pathway in AS fibroblasts. Fibroblasts from hip synovial tissue were isolated, and the expressions of Wnt and Smad pathway proteins were detected by western blot analysis. DKK-1 and SIS3 were used to inhibit Wnt and Smad, respectively. The proliferation and apoptosis of AS fibroblasts were detected by CCK-8 and flow cytometry, respectively. And the effects of DKK-1 and/or SIS3 on osteogenic differentiation of AS fibroblasts were investigated by alkaline phosphatase (ALP) activity, intracellular calcium concentration, mineralization and osteogenic proteins expressions. Results showed that AS fibroblasts had stronger osteogenic differentiation ability, accompanied by activation of Wnt and Smad pathways. In AS fibroblasts, inhibition of Wnt by DKK-1 could affect the Smad pathway, while inhibition of Smad by SIS3 affect the Wnt pathway, DKK-1 and SIS3 showed a synergistic effect on the regulation of Wnt and Smad. In addition, DKK-1 and SIS3 also showed synergistic positive effects in inhibiting the proliferation and osteogenic differentiation of AS fibroblasts. In conclusion, there was an interaction between Wnt and Smad pathways in AS fibroblasts. DKK-1 and SIS3 can synergistically and negatively regulate the osteogenic differentiation of AS fibroblasts, showing positive effects on AS.