The effects of low-magnitude, high-frequency (LMHF) mechanical stimulation on osteoblastic cells are poorly understood. We have developed a system that generates very small (15-40 με), high-frequency (400 Hz, sine) deformations on osteoblast cultures (MC3T3-E1). We investigated the effects of these LMHF stimulations mainly on extracellular matrix (ECM) synthesis. The functional properties of this ECM after decellularization were evaluated on C3H10T1/2 mesenchymal stem cells (MSCs). LMHF stimulations were applied 20 min once daily for 1, 3, or 7 days in MC3T3-E1 culture (1, 3, or 7 dLMHF). Cell number and viability were not affected after 3 or 7 dLMHF. Osteoblast response to LMHF was assessed by an increase in nitric oxide secretion, alteration of the cytoskeleton, and focal contacts. mRNA expression for fibronectin, osteopontin, bone sialoprotein, and type I collagen in LMHF cultures were 1.8-, 1.6-, 1.5-, and 1.7-fold higher than controls, respectively (P < 0.05). In terms of protein, osteopontin levels were increased after 3 dLMHF and ECM organization was altered as shown by fibronectin topology after 7 dLMHF. After decellularization, 7 dLMHF-ECM or control ECM was reseeded with MSCs. Seven dLMHF-ECM improved early events such as cell attachment (2 h) and focal contact adhesion (6 h) and, later (16 h), modified MSC morphological parameters. After 5 days in multipotential medium, gene-expression changes indicated that 7 dLMHF-ECM promoted the expression of osteoblast markers at the expense of adipogenic marker. LMHF stimulations of osteoblasts are therefore efficient and sufficient to generate osteogenic matrix.