Mesenchymal stem cells (MSCs) from bone marrow are a recent source for tissue engineering. Several studies have shown that low-level laser irradiation has numerous biostimulating effects. The purpose of this trial was to evaluate the effects of Nd:Yag laser irradiation on proliferation and differentiation of MSCs induced into the osteoblastic lineage. MSCs were collected from adult human bone marrow, isolated, and cultured in complete medium (α-MEM). Subsequently, they were treated with osteogenic medium, seeded in three-dimensional collagen scaffolds, and incubated. We used six scaffolds, equally divided into three groups: two of these were irradiated with Nd:Yag laser at different power levels (15 Hz, 100 mJ, 1.5 W, and one with a power level of 15 Hz, 150 mJ, 2.25 W), and one was left untreated (control group). Evaluations with specific staining were performed at 7 and 14 days. After 7 days, proliferation was significantly increased in scaffolds treated with laser, compared with the control scaffold. After 14 days, however, laser irradiation did not appear to have any further effect on cell proliferation. As concerns differentiation, an exponential increase was observed after 14 days of laser irradiation, with respect to the control group. However, this was a pilot study with very limited sample size, we conclude, that low-level laser irradiation might lead to a reduction in healing times and potentially reduces risks of failure.