During the aging process, bone marrow mesenchymal stem cells (BMSCs) exhibit declined osteogenesis accompanied by excess adipogenesis, which will lead to osteoporosis. Here, we report that the H3 lysine 36 trimethylation (H3K36me3), catalyzed by histone methyltransferase SET-domain-containing 2 (SETD2), regulates lineage commitment of BMSCs. Deletion of Setd2 in mouse bone marrow mesenchymal stem cells (mBMSCs), through conditional Cre expression driven by Prx1 promoter, resulted in bone loss and marrow adiposity. Loss of Setd2 in BMSCs in vitro facilitated differentiation propensity to adipocytes rather than to osteoblasts. Through conjoint analysis of RNA sequencing (RNA-seq) and chromatin immunoprecipitation sequencing (ChlP-seq) data, we identified a SETD2 functional target gene, Lbp, on which H3K36me3 was enriched, and its expression was affected by Setd2 deficiency. Furthermore, overexpression of lipopolysaccharide-binding protein (LBP) could partially rescue the lack of osteogenesis and enhanced adipogenesis resulting from the absence of Setd2 in BMSCs. Further mechanistic studies demonstrated that the trimethylation level of H3K36 could regulate Lbp transcriptional initiation and elongation. These findings suggest that H3K36me3 mediated by SETD2 could regulate the cell fate of mesenchymal stem cells (MSCs) in vitro and in vivo, indicating that the regulation of H3K36me3 level by targeting SETD2 and/or the administration of downstream LBP may represent a potential therapeutic way for new treatment in metabolic bone diseases, such as osteoporosis.