Alendronate, a nitrogen-containing bisphosphate, is commonly used for the treatment of osteoporosis, showing the potential in enhancing osteogenesis. To improve the osteo-promoting effect of injectable biomineralized microspheres for bone tissue engineering applications, in this study, alendronate was incorporated onto the microspheres during their biomineralization procedure in simulated body fluid (SBF). For the purpose, the microspheres were made of biodegradable poly(L-lactide)-poly(ethylene glycol) block copolymer, which had rough surface with gelatin coating via the (W-1/O)/W-2 emulsion preparation method. Different amounts (25 mu M, 50 mu M or 100 mu M) of alendronate were dissolved in the 5-times SBF and incorporated as calcium minerals depositing onto the microspheres, and thus displaying different release behaviors of alendronate. The cell culture results using bone marrow mesenchymal stromal cells (BMSCs) demonstrated that the microspheres prepared from the 50 mu M of alendronate had the strongest promotion on osteogenic differentiation, while displayed insignificant adverse effect on cell proliferation and viability. By adding Noggin (an inhibitor for bone morphogenetic protein (BMP)) into the culture systems, it was proposed that BMP signaling pathway was one of the possible pathways for alendronate playing its activating effect. In vivo evaluation was conducted by injecting the alendronate-loaded microspheres subcutaneously into rabbit back, in which, histological and immunohistological analysis were done to assess ectopic neovascularization and osteogenesis. Promisingly, the potential of biomineralized microspheres in promoting osteogenesis was significantly strengthened with the incorporation of alendronate. Therefore, a proper application of alendronate in bone repairing materials can be an effective strategy targeting bone regeneration.