Low-level laser irradiation (LLLI) stimulates the proliferation of a variety of cell types. However, very little is known about the effect of laser therapy on dental stem cells. The aim of the present study was to evaluate the effect of LLLI (660 nm, 30 mW) on the proliferation rate of human periodontal ligament stem cells (hPDLSC), obtained from two healthy permanent third molars extracted due to surgical indication. Culture cells were either irradiated or not (control) with an InGaAIP diode laser at 0 and 48 h, using two different energy densities (0.5 J/cm², 16 s and 1.0 J/cm², 33 s). Cell proliferation was evaluated by the Trypan blue exclusion method and by measuring mitochondrial activity using the MTT-based cytotoxicity assay at intervals of 0, 24, 48, and 72 h after the first laser application. An energy density of 1.0 J/cm² improved the cell proliferation in comparison to the other groups (control and laser 0.5 J/cm²) at 48 and 72 h. The group irradiated with 1.0 J/cm² presented significantly higher MTT activity at 48 and 72 h when compared to the energy density of 0.5 J/cm². It can be concluded that LLLI using infrared light and an energy density of 1.0 J/cm² has a positive stimulatory effect on the proliferation of hPDLSC.