Althoughin vivostudies have shown that low-magnitude, high-frequency (LMHF) vibration (LM: < 1 ×g; HF: 20-90 Hz) exhibits anabolic effects on skeletal homeostasis, the underlying cellular/molecular regulation involved in bone adaptation to LMHF vibration is little known. In this report, we tested the effects of microvibration (magnitude: 0.3 ×g, frequency: 40 Hz, amplitude: ± 50 μm, 30 min/12 h) on proliferation and osteodifferentiation of bone marrow-derived mesenchymal stromal cells (BMSCs) seeded on human bone-derived scaffolds. The scaffolds were prepared by partial demineralisation and deproteinisation. BMSCs were allowed to attach to the scaffolds for 3 days. Morphological study showed that spindle-shaped BMSCs almost completely covered the surface of bone-derived scaffold and these cells expressed higher ALP activity than those cultured on plates. After microvibration treatment, BMSC proliferation was decreased on day 7 and 10; however, numbers of genes and proteins expressed during osteogenesis, including Cbfa1, ALP, collagen I and osteocalcin were greatly increased. ERK1/2 activation was involved in microvibration-induced BMSC osteogenesis. Taken together, this study suggests that bone-derived scaffolds have good biocompatibility and show osteoinductive properties. By increasing the osteogenic lineage commitment of BMSCs and enhancing osteogenic gene expressions, microvibration promotes BMSC differentiation and increase bone formation of BMSCs seeded on bone-derived scaffolds. Moreover, ERK1/2 pathway plays an important role in microvibration-induced osteogenesis in BMSC cellular scaffolds.