The effectiveness of CO2 laser therapy in the reduction and elimination of dentinal hypersensitivity in vivo and its thermal effects on tooth surfaces in vitro were investigated. Twenty-three patients with 91 sensitive teeth participated in this study and were followed for 3 months. The parameters used with CO2 laser were 1 W in a continuous wave mode and irradiation time ranging from 5 to 10 s. Hypersensitivity was assessed by thermal stimulus (a blast of air from a dental syringe). Thermal effects were measured by thermography using 10 extracted human teeth. After laser treatment, all patients were immediately free from sensitive pain. Over 3 months, the CO2 laser treatment reduced dentinal hypersensitivity to air stimulus by 50%. All teeth remained vital with no adverse effects. Thermography revealed no temperature increase on irradiated tooth surfaces subjected to water coolant. These results show that the CO2 laser is useful in the treatment of cervical dentinal hypersensitivity without thermal damage to pulp.