Bone tissue engineering is an exciting research area that develops functional strategies for the most challenging bone related clinical issues. Among the numerous materials used in this area, biomimetic materials have been developed amazingly over the past decades. In this study, biomimetic gelatin methacrylamide (Bio-GelMA) hydrogel scaffolds have been fabricated to mimic both the physical architecture and chemical composition of the natural bone extracellular matrix (ECM) by using the thermally induced phase separation (TIPS) technique, to provide three-dimensional templates and extracellular matrix microenvironments. Adipose derived stem cells (ADSCs) also play a pivotal role in osteogenesis when co-cultured with Bio-GelMA at days 7, 14, and 21. The effects of cell-biomaterial interactions such as adhesion, proliferation, and osteogenic differentiation were systematically investigated. The results showed that Bio-GelMA significantly enhanced cell attachment and viability, alkaline phosphatase (ALP) activity, and mineral deposition, as well as mRNA expression levels of osteogenic genes of ADSCs. In a subcutaneous model, H&E staining and dual immunofluorescent staining differed significantly. More importantly, in a critical-size rat calvarial bone defect model, the results of Micro-CT, H&E staining and Masson trichrome staining confirmed that the combination of the Bio-GelMA and ADSCs could enhance osteogenesis significantly. Altogether, the observations prove that the Bio-GelMA scaffolds can act as cell carriers for ADSCs, promote greater osteogenic differentiation of ADSCs and may have great potential in future clinical applications.