Increasing interest has been observed in the use of photobiomodulation (PBM) to enhance the proliferation of stem cells and induce their differentiation. The effects of PBM at two different wavelengths (635 and 809 nm) with three different energy densities (0.5, 1 and 2 J/cm) on the osteogenic differentiation of adipose-derived stem cells (ADSC) were investigated. Cell viability and proliferation were evaluated by MTT and Alamar Blue assays. Osteoblast differentiation were assessed by alkaline phosphatase (ALP) activity, Alizarin red staining and reverse-transcription polymerase chain reaction (RT-PCR) for the expression of collagen type I (COL1A), ALP and osteocalcin. 635 nm and 809 nm laser irradiation had no effect on the cell viability on days 7 and 14, except for 0.5 J/cm group at 14th day after 635 nm irradiation (p < 0.05). Cell proliferation was not changed significantly. Mineralization was increased significantly in 809 nm laser groups but no enhancement was detected in the osteogenic differentiation by ALP activity and gene expression results. In 0.5 and 1 J/cm groups, ALP and COL1A expressions were down regulated at day 7 after 809 nm laser exposure. These results suggest that PBM may alter osteogenic differentiation of ADSC and increase mineralization but further investigation is needed to define adequate parameters.