Objective: Distal-less homeobox 3 (DLX3) is an important transcription factor involved in the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). However, the underlying mechanism is not clear. This study investigated the underlying mechanism of DLX3 in osteogenic differentiation. Methods: DLX3 overexpression and knockdown in cells were achieved using lentiviruses. The osteogenic differentiation of BMSCs was detected using alkaline phosphatase expression, alizarin red staining, real-time quantitative polymerase chain reaction (RT-qPCR), Western blotting, and chromatin immunoprecipitation (ChIP) assays. Results: DLX3 overexpression promoted the osteogenic differentiation of BMSCs, whereas DLX3 knockdown reduced the osteogenic differentiation of BMSCs. RT-qPCR and Western blotting assays showed that DLX3 modulated osteogenic differentiation via the Wnt/beta-catenin pathway. ChIP-qPCR showed that DLX3 knockdown promoted DKK4 expression by decreasing the enrichment of histone H3 lysine 27 trimethylation (H3K27me3) in the promotor region of DKK4. Conclusion: Our data implied that DLX3 regulated Wnt/beta-catenin pathway through histone modification of DKK4 during the osteogenic differentiation of BMSCs. (C) 2019 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).