Retaining or improving periodontal ligament (PDL) function is crucial for restoring periodontal defects. The aim of this study was to evaluate the physiological effects of low-power laser irradiation (LPLI) on the proliferation and osteogenic differentiation of human PDL (hPDL) cells. Cultured hPDL cells were irradiated (660 nm) daily with doses of 0, 1, 2 or 4 J·cm-2 . Cell proliferation was evaluated by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay, and the effect of LPLI on osteogenic differentiation was assessed by Alizarin Red S staining and alkaline phosphatase (ALP) activity. Additionally, osteogenic marker gene expression was confirmed by real-time reverse transcription-polymerase chain reaction (RT-PCR). Our data showed that LPLI at a dose of 2 J·cm-2 significantly promoted hPDL cell proliferation at days 3 and 5. In addition, LPLI at energy doses of 2 and 4 J·cm-2 showed potential osteogenic capacity, as it stimulated ALP activity, calcium deposition, and osteogenic gene expression. We also showed that cyclic adenosine monophosphate (cAMP) is a critical regulator of the LPLI-mediated effects on hPDL cells. This study shows that LPLI can promote the proliferation and osteogenic differentiation of hPDL cells. These results suggest the potential use of LPLI in clinical applications for periodontal tissue regeneration. The aim of this study was to evaluate the physiological effects of low-power laser irradiation (LPLI) on the proliferation and osteogenic differentiation of human PDL (hPDL) cells. Cultured hPDL cells were irradiated (660 nm) daily with doses of 0, 1, 2 or 4 J · cm -2. Cell proliferation was evaluated by the 3- [4,5-dimethylthiazol-2-yl] diphenyltetrazolium bromide (MTT) assay, and the effect of LPLI on osteogenic differentiation was assessed by Alizarin Red S staining and alkaline phosphatase (ALP) activity. Additionally, osteogenic marker gene expression was confirmed by real-time reverse transcription- polymerase chain reaction -PCR). Our data showed that LPLI at a dose of 2 J · cm -2 showed promoted hPDL cell proliferation at days 3 and 5. In addition, LPLI at energy doses of 2 and 4 J · cm-2 showed potential osteogenic capacity , as it stimulated ALP activity, calcium de position, and osteogenic gene expression. We also showed that cyclic adenosine monophosphate (cAMP) is a critical regulator of the LPLI-mediated effects on hPDL cells. This study suggests that LPLI can promote the proliferation and osteogenic differentiation of hPDL cells. the potential use of LPLI in clinical applications for periodontal tissue regeneration.