Visible light irradiation is an emerging area in regenerative medicine research. We hypothesized that low-intensity-pulsed LED light irradiance may exert photobiomodulatory effects on cultured osteoblast-like cells. To test this hypothesis, we investigated cell proliferation and markers of cell maturation and metabolic activity following pulsed LED irradiance. Monolayer explant cultures of human osteoblast-like cells were exposed four times in 24-h intervals to 2 min of pulsed white LED irradiance of 2.4-2.5 mWÁcm À2 and its different spectra of 0.2-0.5 mWÁcm À2 (frequency range of 10-40 Hz). Cell proliferation was estimated from microscopic cell counting and cell death by lactate dehydrogenase activity in culture media (measured by a colorimetric method). The early markers of osteoblast maturation and metabolic activity, that is, cellular alkaline phosphatase activity and osteocalcin content, were measured using a colorimetric method and ELISA, respectively. Irradiance of 40 Hz caused the highest increase in cell number (P < 0.01). Osteocalcin content in cells decreased following 40 Hz and 10 Hz irradiance (P < 0.05). The 40 Hz blue range irradiance (diffuse transmittance 420-580 nm, maximal cell irradiance 0.5 mWÁcm À2) caused a decrease in alkaline phosphatase cellular activity (P < 0.001) and an increase in media osteocalcin content (P < 0.05). The 40 Hz green range (diffuse transmittance 560-650 nm, maximal cell irradiance 0.4 mWÁcm À2) irradiance caused an increase in the number of cells and in cell death. In summary, pulsed (40 Hz) white light irradiance has photomodulatory effects, with its green range spectrum affecting cell proliferation and cell death, and its blue range spectrum affecting cellular maturation and metabolism. The results indicate a low-intensity threshold of photobiomodulation of osteoblast-like cells in vitro.