Background The field of laser-based photochemical reactions receives a great promising for additional applications especially for targeting cells, pathogens, or molecules. Limited studies have investigated the impact of light-emitting diode on stem cell behavior. Thus, the aim of the present study was to assess the effect of diode laser irradiations on the proliferation of stem cells isolated from the human dental pulp. Isolation procedures were according to previously developed protocols for dental pulp stem cells (DPSCs). Low-level laser therapy irradiation (LLLT) was applied in two doses (0.5 J/cm2 and 1 J/cm2 for 20 s) into 96-well plates by the diode laser device (970 nm) through the fiber optic (SiroLaser fibers 320) at a distance from the opening of the wells to be accurate for equal distribution of the laser irradiation. To assess the proliferation capacity of the isolated stem cells, MTT assay was performed 24 h, 48 h, and 72 h. Results There was no significant difference among the different groups (gp 1 control, gp II dose 0.5 J/cm2, gp III dose 1 J/cm2) on day 1. While on day 2, the optical density of DPSCs subjected to dose 1 was found to be significantly higher than that of those subjected to dose 2. This was also demonstrated on day 3. It was also demonstrated that the proliferation of DPSCs subjected to dose 1 increased compared to that of DPSCs subjected to dose 2 from day 1 to day 2. There was a significant decrease in the cell number in both groups by 72 h. Conclusion In conclusion, the use of LLLT as a stimulatory factor for enhancing and proliferation of the stem cells is very promising.