Bone loss due to osteoporosis or disuse such as in paraplegia or microgravity is a significant health problem. As a treatment for osteoporosis, brief exposure of intact animals or humans to low magnitude and high frequency (LMHF) mechanical loading has been shown to normalize and prevent bone loss. However, the underlying molecular changes and the target cells by which LMHF mechanical loading alleviate bone loss are not known. Here, we hypothesized that direct application of LMHF mechanical loading to osteoblasts alters their cell responses, preventing decreased bone formation induced by disuse or microgravity conditions. To test our hypothesis, preosteoblast 2T3 cells were exposed to a disuse condition using the random positioning machine (RPM) and intervened with an LMHF mechanical load (0.1-0.4 g at 30 Hz for 10-60 min/day). Exposure of 2T3 cells to the RPM decreased bone formation responses as determined by alkaline phosphatase (ALP) activity and mineralization even in the presence of a submaximal dose of BMP4 (20 ng/ml). However, LMHF mechanical loading prevented the RPM-induced decrease in ALP activity and mineralization. Mineralization induced by LMHF mechanical loading was enhanced by treatment with bone morphogenic protein 4 (BMP4) and blocked by the BMP antagonist noggin, suggesting a role for BMPs in this response. In addition, LMHF mechanical loading rescued the RPM-induced decrease in gene expression of ALP, runx2, osteomodulin, parathyroid hormone receptor 1, and osteoglycin. These findings suggest that preosteoblasts may directly respond to LMHF mechanical loading to induce differentiation responses. The mechanosensitive genes identified here provide potential targets for pharmaceutical treatments that may be used in combination with low level mechanical loading to better treat osteoporosis or disuse-induced bone loss.