We investigated the effect of shockwaves on cells explanted from normal human periosteum to study the potential mechanisms of their responses and to determine suitable treatment settings. The cells were subjected to one shockwave treatment with systematic combinations of energy intensities (range, 0.05-0.5 mJ/mm) and number of shocks (range, 500-2000) whereas control cells received no treatment. The immediate effect on cell viability and the long-lasting effect on proliferation, viable cell number at Day 18, and mineralization at Day 35 were assessed. We observed an immediate dose-dependent destructive effect of shockwaves. Energy intensity and number of shocks contributed equally to viability. Total energy dose (intensity x number of shocks) was a better reference for determining the shockwave effect. We also found a long-term stimulatory effect on proliferation, viable cell number, and calcium deposition of human periosteal cells. At the same total energy dose, low-intensity shockwaves with more shocks (0.12 mJ/mm at 1250 shocks) were more favorable for enhancing cellular activities than high-intensity waves with fewer shocks (0.5 mJ/mm at 300 shocks). These findings document some of the biochemical changes of periosteal cells during shockwave treatments.