BACKGROUND: Fracture is one of the most pervasive injuries in the musculoskeletal system, and there is a complex interaction between macrophages and adipose tissue-derived stem cells (ADSCs) in fracture healing. However, two-dimensional (2D) coculture of macrophages and ADSCs can not accurately mimic the AIM: To establish both 2D and 3D osteogenic coculture models to investigate the interaction between macrophages and ADSCs. METHODS: After obtaining ADSCs from surgery and inducing differentiation of the THP1 cell line, we established 2D and 3D osteogenic coculture models. To assess the level of osteogenic differentiation, we used alizarin red staining and measured the relative expression levels of osteogenic differentiation markers osteocalcin, Runt-related transcription factor 2, and alkaline phosphatase through polymerase chain reaction. Verification was conducted by analyzing the expression changes of N-cadherin and the activation of the Wnt/β-catenin signaling pathway using western blotting. RESULTS: In this study, it was discovered that macrophages in 3D culture inhibited osteogenic differentiation of ADSCs, contrary to the effect in 2D culture. This observation confirmed the significance of intricate intercellular connections in the 3D culture environment. Additionally, the 3D culture group exhibited significantly higher N-cadherin expression and showed reduced β-catenin and Wnt1 protein levels compared to the 2D culture group. CONCLUSION: Macrophages promoted ADSC osteogenic differentiation in 2D culture conditions but inhibited it in 3D culture. The 3D culture environment might inhibit the Wnt/β-catenin signaling pathway by upregulating N-cadherin expression, ultimately hindering the osteogenic differentiation of ADSCs. By investigating the process of osteogenesis in ADSCs, this study provides novel ideas for exploring 3D osteogenesis in ADSCs, fracture repair, and other bone trauma repair.