Extracorporeal shock wave therapy has been used increasingly in musculoskeletal disorders although its biologic mechanisms are not understood completely. The current study evaluated the effects of extracorporeal shock wave therapy on human osteoblastlike cells by using an electrohydraulic shock wave generator and comparing three energy levels. (Group A, 14 kV and 0.15 mJ/mm2; Group B, 21 kV and 0.31 mJ/mm2; Group C, 28 kV and 0.40 mJ/mm2; Control Group, no energy) and two total impulses (500, 1000) for each level. At the end of treatment, a reduction by approximately 76% was observed in Group C cell number versus basal value when compared with the other groups. Viability, biochemical activity, and gene expression of cultured cells were evaluated 24 and 48 hours after treatment. The viability test showed a decrease in Group C viability of approximately 54% at both culture times as compared with the other groups. Significant increases in nitric oxide, osteocalcin, and transforming growth factor-beta1 production ranging from 10% to 35% were found in Group A. All treated groups had lower C-terminal procollagen Type I values than the Control Group, but important increases were observed between 24 and 48 hours in all groups except Group C. This particular finding reveals that osteoblast differentiation in Group A is enhanced strongly during the first 24 hours after exposure leading after another 24 hours to an increase in C-terminal procollagen Type I production and consequently in bone matrix deposition. The current study showed that one of the most important aspects to be considered is not the total number of impulses used, but the energy level of the shock waves, therefore confirming that extracorporeal shock wave therapy has a dose-dependent initial destructive effect on cells when the selected energy is higher than 21 kV.