Several methods have been proposed to enhance the regeneration and healing time in periodontal therapy. Photobiomodulation therapy (PBMT) is a recently suggested novel technique for this purpose. This study aimed to compare the efficacy of PBMT with various laser wavelengths and energy densities on proliferation of human periodontal ligament mesenchymal stem cells (PDLMSCs). The wells containing PDLMSCs were subjected to laser irradiation at 635, 660, 808, and 980 nm wavelengths with 1, 1.5, 2.5 and 4 J/cm2 energy densities. Cell proliferation and viability were evaluated after 1, 3, and 5 days with the methyl thiazolyl tetrazolium (MTT) assay and 4,6-diamidino-2-phenylindole (DAPI) staining. No significant difference was observed among the experimental and the control groups on day 1 (P>0.05). On day 3, 808 nm laser at 4 J/cm2 energy density and 980 nm laser at all densities had significant differences with control group. On day 5, the control group had significant differences in cell proliferation with 808 nm laser at 2.5 and 4 J/cm2 energy densities, and 980 nm laser at all densities. PBMT with 635, 660, 808, and 980 nm wavelengths increased the proliferation of PDLMSCs but the maximum cell viability was prominent after irradiation by 980 nm laser with energy density of 4 J/cm2 on day 3.