Photobiomodulation (PBM) using red or near-infrared (NIR) light has been used to stimulate the proliferation and differentiation of adipose-derived stem cells. The use of NIR wavelengths such as 810nm is reasonably well accepted to stimulate mitochondrial activity and ATP production via absorption of photons by cytochrome c oxidase. However, the mechanism of action of 980nm is less well understood. Here we study the effects of both wavelengths (810nm and 980nm) on adipose-derived stem cells in vitro. Both wavelengths showed a biphasic dose response, but 810nm had a peak dose response at 3J/cm for stimulation of proliferation at 24h, while the peak dose for 980nm was 10-100 times lower at 0.03 or 0.3J/cm. Moreover, 980nm (but not 810nm) increased cytosolic calcium while decreasing mitochondrial calcium. The effects of 980nm could be blocked by calcium channel blockers (capsazepine for TRPV1 and SKF96365 for TRPC channels), which had no effect on 810nm. To test the hypothesis that the chromophore for 980nm was intracellular water, which could possibly form a microscopic temperature gradient upon laser irradiation, we added cold medium (4°C) during the light exposure, or pre-incubated the cells at 42°C, both of which abrogated the effect of 980nm but not 810nm. We conclude that 980nm affects temperature-gated calcium ion channels, while 810nm largely affects mitochondrial cytochrome c oxidase.