Diffuse idiopathic skeletal hyperostosis (DISH) is a noninflammatory skeletal disease characterized by the progressive ectopic ossification and calcification of ligaments and enthuses. However, specific pathogenesis remains unknown. Bone marrow mesenchymal stem cells (BMSCs) are a major source of osteoblasts and play vital roles in bone metabolism and ectopic osteogenesis. However, it is unclear whether BMSCs are involved in ectopic calcification and ossification in DISH. The current study aimed to explore the osteogenic differentiation abilities of BMSCs from DISH patients (DISH-BMSCs). Our results showed that DISH-BMSCs exhibited stronger osteogenic differentiation abilities than normal control (NC)-BMSCs. Human cytokine array kit analysis showed significantly increased secretion of Galectin-3 in DISH-BMSCs. Furthermore, Galectin-3 downregulation inhibited the increased osteogenic differentiation ability of DISH-BMSCs, whereas exogenous Galectin-3 significantly enhanced the osteogenic differentiation ability of NC-BMSCs. Notably, the increased Galectin-3 in DISH-BMSCs enhanced the expression of β-catenin as well as TCF-4, whereas attenuation of Wnt/β-catenin signaling partially alleviated Galectin-3-induced osteogenic differentiation and activity in DISH-BMSCs. In addition, our results noted that Galectin-3 interacted with β-catenin and enhanced its nuclear accumulation. Further in vivo studies showed that exogenous Galectin-3 enhanced ectopic bone formation in the Achilles tendon in trauma-induced rats by activating Wnt/β-catenin signaling. The current study indicated that enhanced osteogenic differentiation of DISH-BMSCs was mainly attributed to the increased secretion of Galectin-3 by DISH-BMSCs, which enhanced β-catenin expression and its nuclear accumulation. Our study helps illuminate the mechanisms of pathological osteogenesis and sheds light on the possible development of potential therapeutic strategies for DISH treatment.