Ankylosing spondylitis (AS) refers to a type of arthritis manifested with chronic inflammation of spine joints. microRNAs (MiRNAs) have been identified as new therapeutic targets for inflammatory diseases. In this study, we evaluated the influence of microRNA‐96 (miR‐96) on osteoblast differentiation together with bone formation in a murine model of AS. The speculated relationship that miR‐96 could bind to sclerostin (SOST) was verified by dual luciferase reporter assay. After successful model establishment, the mice with AS and osteoblasts isolated from mice with AS were treated with mimics or inhibitors of miR‐96, or DKK‐1 (a Wnt signaling inhibitor). The effects of gain‐ or loss‐of‐function of miR‐96 on the inflammatory cytokine release (IL‐6, IL‐10, and TNF‐α), alkaline phosphatase (ALP) activity, calcium nodule formation, along with the viability of osteoblasts were determined. It was observed that miR‐96 might target and regulate SOST. Besides, miR‐96 was expressed at a high level in AS mice while SOST expressed at a low level. TOP/FOP‐Flash luciferase reporter assay confirmed that miR‐96 activated the Wnt signaling pathway. Moreover, AS mice overexpressing miR‐96 exhibited increased contents of IL‐6, IL‐10 and TNF‐α, ALP activity, calcium nodule numbers, and viability of osteoblasts. In contrast, inhibition of miR‐96 resulted in suppression of the osteoblast differentiation and bone formation. In conclusion, the study implicates that overexpressing miR‐96 could improve osteoblast differentiation and bone formation in AS mice via Wnt signaling pathway activation, highlighting a potential new target for AS treatment.