Lithium have been shown to play an important role in improving the osteogenic properties of biomaterials. This study aims to explore the osteogenic improvement effect of tissue engineered heterogeneous deproteinized bone (HDPB) doped with lithium, and evaluate their effectiveness in the healing of bone defects. Bone marrow mesenchymal stem cells (BMSCs) were co-cultured with different concentration of lithium chloride. Cell proliferation in each group was analyzed by 3-(4, 5-dimetyl-2-thiazoly-2, 5-diphenyl-2-H-tetrazolium bromide (MTT) assay. BMSCs were then co-cultured in osteogenic induction medium with different concentration of lithium chloride, and the expression of related mRNA was detected. The role of lithium in promoting BMSCs osteogenic differentiation and inhibiting BMSCs lipogenic differentiation was also investigated. Biomechanical properties of the tibia were evaluated at 8 weeks after operation. The tibial specimens of each group were collected at 4 and 8 weeks after surgery for histological examination and histological analysis. Micro-computed tomography (CT) scanning and 3D reconstruction were performed at 8 weeks. The results demonstrate that lithium can induce the osteogenic differentiation inhibit of adipogenic differentiation of BMSCs by regulating the Wnt signaling pathway. The histological evaluation further certified that average bone formation area in the group of tissue engineered HDPB doped with lithium was also significantly better than that of HDPB alone group. Based on the above evaluation, tissue engineered HDPB doped with lithium can effectively promote the regeneration of segmental bone defect, which can be used as a tissue engineering scaffold for clinical trials.