It is suggested that pulpal fluid circulation has an impact on pulp temperature increase during heat-generating dental treatment procedures. Thus, the aim of the study was to assess the effect of a simulated pulpal fluid circulation on temperature changes inside the pulp chamber following laser irradiation of the tooth surface. Twenty freshly extracted human multirooted teeth were included and cross-sectioned along the long axis exposing two root canals each. The pulp chamber and root canals were cleaned from remaining soft tissues to achieve access for a temperature sensor and two cannulas to allow fluid circulation. Cross sections were glued together, and the roots were encased with silicone impression material to ensure the position of the connected devices. Each tooth was irradiated by employing a neodymium-doped yttrium orthovanadate (Nd:YVO4) laser at 1,064 nm with a pulse duration of 9 ps and a repetition rate of 500 kHz. A commercially available scanning system (SCANcube 7, SCANLAB) deflected the beam by providing rectangular irradiated areas of 0.5 mm edge length. Measurements were performed with four different settings for fluid circulation: without any water and with water (23 °C) at a flow rate of 6, 3, and 0 ml/min. The primary outcome measure was the maximum temperature difference (ΔT) after laser irradiation. Highest temperature changes (median 3.6 K, range 0.5-7.1 K) could be observed without any fluid inside the pulp chamber. Water without circulation decreased ΔT values statistically significantly (median 1.4 K, range 0.2-4.9 K) (p < 0.05). Lowest temperature changes could be observed with a water flow rate of 6 ml/min (median 0.8 K, range 0.2-3.7 K) (p < 0.05). Pulpal fluid circulation has a cooling effect on temperature increase caused by laser irradiation of dental hard tissues. Studies on heat generation during dental treatment procedures should include this aspect to assess a potential thermal injury of pulp tissue.