Whereas mechanical stimulation is essential for bone homeostasis, straining of larger magnitude promotes bone regeneration by directing cell differentiation and proliferation and influencing the gene expression patterns of osteoblasts, which play a vital role in fracture healing by producing and mineralizing osteoid matrix. To elucidate the molecular mechanisms underlying the response of osteoblasts to mechanical strains comparable to those occurring during bone regeneration, MC3T3 S4 (MC4) osteoblast-like cells were stretched in vitro. Analysis based on microarray expression profiling during the first 8 h after straining showed 674 differentially expressed genes. The response to mechanical strain can be divided in an immediate-early response (IER) and later responses. Examination of the approximately 40 genes differentially expressed within the first 60 min, including 11 involved in regulating gene transcription, showed both promiscuous IER genes such as Fos that are upregulated by multiple extracellular stimuli, as well as a number of genes previously shown in neurons to be induced preferentially by depolarization (IPD-IER). Selected differentially expressed genes were validated after mechanical straining and KCl-induced depolarization. The effects of inhibitors for protein kinase A, mitogen-activated protein kinase, and calcineurin pathways were assessed in separate experiments by quantitative RT-PCR and shown to have differential effects on the response of MC4 cells and primary calvaria osteoblasts to both mechanical straining and KCl-induced depolarization. Therefore, our results showed the existence of two distinct pathways that mediate the IER of osteoblasts to large-magnitude mechanical straining and suggest that the IER to depolarizing stimuli is conserved in cell types as different as osteoblasts and neurons.