Despite the increasing use of lasers in the rehabilitation of horses, the biophysical action of the laser is not clearly defined. The purpose of this study was to determine the effect of a defocused CO2 laser on the temperature of the skin, subcutis and fetlock joint in standing and anaesthetized horses. A cross-over design comprising 10 standing horses was used. Consecutive irradiation (91 J cm−2) was applied to each of the three aspects of the front fetlock joint of these animals. In 12 anaesthetized horses (eight laser-treated and four control), irradiation (137 J cm−1) was applied to the dorsal side of the joint. In the standing group, skin temperature increased on average by 5.3°C to 34.8±1.5° (P<0.05) and the subcutis temperature increased by 5.7°C to a mean temperature of 36.0±0.9°C during laser treatment. There was no difference in joint temperature between laser-treated and control horses. Similar results were obtained in anaesthetized horses. Treatment with a defocused CO2 laser caused a significant increase in the temperature of the skin and subcutis, but not in the joint cavity. Further studies are needed to investigate if the increase in temperature influences perfusion and modulation of pain, as a result of defocused CO2 laser treatment.