Summary Background Administration of pulsed inhaled nitric oxide (PiNO) improves arterial oxygenation in spontaneously breathing anaesthetised healthy horses and in horses undergoing colic surgery. However, because hypoventilation commonly occurs, horses are often mechanically ventilated to prevent hypercarbia. Objectives To evaluate the effects of PiNO on arterial oxygenation during anaesthesia in mechanically ventilated healthy horses and horses undergoing colic surgery. Study design Prospective nonblinded clinical trial. Methods Fifty horses undergoing elective arthroscopy (Group A) and 30 horses undergoing colic surgery (Group C) in dorsal recumbency were included in the study. Every second horse in each group received PiNO (A‐INO, C‐INO), the others served as controls (A‐CN, C‐CN). All horses were mechanically ventilated and anaesthesia was maintained with isoflurane. PiNO was mechanically delivered at the proximal end of the endotracheal tube as a pulse during the first part of each inspiration. Data were collected at the start (baseline, before PiNO) and at the end of inhalation anaesthesia. The Tukey method was used to compare baseline and end values for each parameter. Results Arterial oxygen tension (PaO2) increased from (median [IQR]) 13.6 (9.3, 30.1) at baseline to 24.2 (18.6, 37.0) kPa at the end of anaesthesia in A‐INO (P = 0.005) and from 7.7 (6.4, 8.5) to 15.5 (9.9, 26.9) kPa in C‐INO (P = 0.007). Mean (95% CI) difference in F‐shunt between baseline and end were −6 (−10; −1) and −11 (−22; −1) % in A‐INO (P = 0.005) and C‐INO (P = 0.04) respectively. There was no change in PaO2 or F‐shunt from baseline to end of anaesthesia in A‐CN or C‐CN. Main limitations Cardiac output was not measured, thus O2 delivery could not be calculated. Conclusions The combination of mechanical ventilation and PiNO improved pulmonary gas exchange during anaesthesia by a simultaneous decrease in F‐shunt and improved alveolar ventilation.