Calcinated antler cancellous bone (CACB) is useful in repair of bone defects, as its composition and architecture is analogous to natural extracellular bone matrix. The use of CACB scaffolds with adipose-derived stem cells (ADSCs) in repair of rabbit mandibular bone defects was investigated along with the underlying molecular signaling pathways involved. CACB promoted the adhesion, spreading, and viability of ADSCs. Increased extracellular matrix production and expression of osteogenic markers in ADSCs were observed when seeded in CACB. The temporal kinetics of mRNA expression of ADSCs cultured in CACB lagged in comparison with that observed in cells grown in medium with osteogenic supplements. Activation of the extracellular signal-related kinases (ERK) 1/2 and RUNX-2 in CACB-cultured ADSCs was observed, and this activation was attenuated by the MeK inhibitor U0126. Microcomputed tomography scanning analysis and histological evaluations showed that loading the CACB with ADSCs resulted in enhanced new bone formation and angiogenesis when the composites were implanted in rabbit mandibular defects. These results indicated that the osteogenic behavior of ADSCs might be driven by the microenvironment formed by CACB via the ERK signaling pathway. These CACB/ADSCs composites have promising therapeutic potential for large bone defect repairs.