Bushen-Qiangdu-Zhilv (BQZ) decoction is a traditional Chinese medicinal compound widely used for treating ankylosing spondylitis (AS). However, the mechanisms underlying effects of BQZ remain largely unknown. Osteoblast differentiation of fibroblasts plays an important role in heterotopic ossification (HO) of AS, and connexin 43 (Cx43) is crucially involved in the osteoblast differentiation of fibroblasts. The aim of the present study was to evaluate the effects of BQZ on the osteogenic differentiation of fibroblasts by regulating Cx43. Rat fibroblasts were treated with freeze-dried powder of BQZ, in the presence or absence of recombinant human bone morphogenetic protein-2 (rhBMP-2). MTS assays were performed to examine the inhibitory effects of BQZ on fibroblast proliferation. Western blot assays were conducted to detect the protein expression of core-binding factor alpha 1 (Cbfa1), Cx43 and phosphorylated Cx43 (pCx43). BQZ appeared to inhibit fibroblast proliferation in a dose-dependent manner. Furthermore, the expression of Cbfa1 and Cx43/pCx43 was significantly suppressed by BQZ, with or without rhBMP-2 stimulation. Therefore, the present results indicate that BQZ may exert an anti-AS effect by suppressing the osteogenic differentiation of fibroblasts via Cx43 regulation.