Dentin hypersensitivity (DH) is one of the most common complications that affect patients after periodontal therapy. So far, many investigators have successfully used different types of laser on DH treatment. The aim of this study was to evaluate the comparative effect of Nd:YAG laser and Er:YAG laser on human teeth desensitization. A group of nine patients with a total of 63 chronic hypersensitive teeth were selected. Each one of them should at least have three hypersensitive teeth. These teeth were randomly allocated into three groups. Group 1, Nd:YAG laser (1 W, 15 Hz, 60 s, two times); group 2, Er:YAG laser (100 mJ, 3 Hz, 60 s, two times); and group 3 serves as control group without any treatment. Assessment of pain was performed by a visual analysing scale (VAS) after stimulation of sensitive tooth by using the sharp tip of an explorer. This test was performed before treatment, immediately after that and at 1-, 3- and 6-month intervals after treatment by one blinded examiner. Analysis of VAS score between the three groups at the time of treatment did not show any significant difference (p = 0.506). However, by using repeated-measurement analysis of variance test, significant differences were seen in the three groups between before-treatment VAS score and after treatment (p < 0.0005). This statistically significant difference in the control group demonstrated a placebo effect. However, the effect of using Nd:YAG and Er:YAG lasers was stronger than this placebo effect, so that after removing the effect of the placebo, differences immediately after, 1, 3 and 6 months post treatment between all three groups still were statistically highly significant (p < 0.0005). Compared to the Er:YAG laser group, using Nd:YAG laser resulted in a significant reduction of VAS score at each follow-up examination (p < 0.0005). Although using Nd:YAG and Er:YAG laser in desensitization of hypersensitive teeth showed a placebo effect limited to a short time, results of this study demonstrated that both of these lasers have an acceptable therapeutic effect. The observed effects seemed to last for at least 6 months. It was concluded that Nd:YAG laser is more effective than Er:YAG laser in reduction of patients' pain.