Low-level laser therapy (LLLT) is widely used in regenerative medicine and in dental therapy by virtue of its beneficial effects in a plethora of pathological conditions. In this study, the effect of a 980 nm diode laser on pre-osteoblasts proliferation has been evaluated, along with reactive oxygen species (ROS) production. We hypothesized that ROS were a key factor in LLLT-induced pre-osteoblasts proliferation, as it is known that ROS can induce the activation of many biological pathways, leading to cell proliferation, differentiation or apoptosis. Murine pre-osteoblasts MC3T3 cells were irradiated with different energy outputs (1-50 J) in the absence or presence of the antioxidant N-Acetyl-L-cysteine (NAC). Laser treatment, in the absence of NAC, was able to induce a fluence-dependent statistically significant increase in ROS generation, while the presence of NAC strongly inhibited it. Cell proliferation, measured after laser stimulation, was significantly increased both at low and higher energy, with a peak at 10 J in the absence of the antioxidant. On the contrary, in the presence of NAC, laser irradiation was not able to induce any cell proliferation, suggesting a crucial role of ROS in this laser-induced cell effect. These results suggest that LLLT may be a useful tool for bone regeneration therapy and an effective range of fluences to be used is indicated.