Objective Mesoporous bioactive glass (MBG) has good biocompatibility without immune reaction after implanting into tissue as biomaterial which was used in the treatment of bone defect. Genistein (G), a phytoestrogen, could be used in the treatment of osteoporosis in postmenopausal women. Results Here, we report that MBG with large pores (MBG-L) and MBG-L adsorbed with G (MBG-L/G) sustained-release G could enhance osteoblast differentiation and matrix mineralization. Interestingly, we observed that MBG-L enhanced the formation of bone-like deposit and Ca deposition in vitro. In the other side, we also found that MBG-L/G substrate could promote osteoblast differentiation and matrix mineralization through Erk activated Runx2 pathway. Interestingly, the expression of osteoblast-specific marker gene Osteopontin (Opn) was also increased in MC3T3-E1 cells cultured on MBG-L/G substrate. Conclusions We conclude that MBG-L/G is a potential biomaterial for the treatment of bone defect.