Whole body vibration (WBV), consisting of a low-magnitude, high-frequency (LMHF) signal, is anabolic to bone in vivo and may act through alteration of the lineage commitment of mesenchymal stromal cells (MSC). We investigated the effect of LMHF vibration on rat bone marrow-derived MSCs (rMSCs) in an in vitro system. We subjected rMSCs to repeated (six) bouts of 1-h vibration at 0.3g and 60 Hz in the presence of osteogenic (OS) induction medium. The OS differentiation of rMSCs under the loaded and non-loaded conditions was assessed by examining cell proliferation, alkaline phosphatase (ALP) activity, mRNA expression of various osteoblast-associated markers [ALP, Runx2, osterix (Osx), collagen type I alpha 1 (COL1A1), bone sialoprotein (BSP), osteopontin (OPN), and osteocalcin (OCN)], and matrix mineralization. LMHF vibration did not enhance the OS differentiation of rMSCs. Surprisingly, the mRNA level of Osx, a transcription factor necessary for osteoblast formation, was decreased, and matrix mineralization was inhibited. Our findings suggest that LMHF vibration may exert its anabolic effects in vivo via mechanosensing of a cell type different from MSCs.