The application of Nd:YAG lasers in endodontics has been explored by many investigators for different purposes. The aim of this in vitro study is to evaluate the effects of Nd:YAG laser on osteoblast cell cultures. A 1,064 nm Nd:YAG laser was used to irradiate human osteoblast-like cells (Saos-2) in noncontact mode for 10 s with different energy settings. The settings varied were pulse energy (20-120 mJ), pulse repetition rate (10-30 Hz), and power output (0.2-3.6 W). He-Ne, the aiming beam of Nd:YAG laser, was used in noncontact mode for 10 s. After 7-, 14-, and 21-day incubation period, cell viability and proliferation were evaluated with methylthiazoletetrazolium (MTT) colorometric method. The comparison of average MTT values gave no statistically significant difference between 20 mJ, 10 Hz, He-Ne, and control (p < 0.05). The differences between all other groups were significant (p < 0.05). Increase in the pulse energy, pulse repetition rate, and power output has a negative effect on the cell viability and proliferation.