Human acellular amniotic membrane is an acellular, naturally extracellular matrix material with various bioactive factors, which applied in tissue engineering in clinic. Several studies have applied human acellular amniotic membrane in skin and ocular surface tissue engineering to enhance tissue regeneration. However, the application of human acellular amniotic membrane in bone tissue engineering was rarely investigated. The aim of the current study was to investigate the osteoinductivity, angiogenesis and the early molecular changes of human acellular amniotic membrane to bone regeneration. Our results showed that human acellular amniotic membrane with excellent biocompatibility was beneficial for bone marrow mesenchymal stem cells proliferation and osteogenic differentiation. In rat femoral defect model, the existence of human acellular amniotic membrane significantly improved bone regeneration in the defects. The gene expression of CXCR-4, MCP-1, OC and CatK which were connected with cells recruitment and bone remodeling, was enhanced in the defects implanted with human acellular amniotic membrane. The results of this study suggest that human acellular amniotic membrane is an osteoinductive biomaterial for bone regeneration.