Acute respiratory distress syndrome (ARDS) is a clinical syndrome characterized by refractory hypoxemia resulting from ventilation-perfusion (V/Q) mismatch. Studies have shown that inhaled nitric oxide (iNO) and prone position (PP) ventilation may improve V/Q mismatch and oxygenation when used separately. Despite the known individual benefits of iNO and PP, few studies have investigated their potential synergistic effects. The aim of this study was to evaluate iNO combined with PP on V/Q matching and oxygenation in patients with moderate-to-severe ARDS.A 2 × 2 factorial design was adopted in this study. Patients admitted to the Intensive Care Unit of Northern Jiangsu People's Hospital from January 2024 to December 2024, who met the diagnostic criteria for moderate-to-severe ARDS (aged 18-80 years, oxygenation index <150 mmHg, and requiring mechanical ventilation), were enrolled. The patients were administered with a combination of iNO therapy at 20 ppm while in the supine position (SP) and prone position (PP). Various clinical variables were collected at baseline in the SP, as well as at 4 and 12-h during PP, and after 30-60 min of iNO treatment (SP, SP + iNO, PP, PP + iNO). The pulmonary ventilation-perfusion status, such as the global inhomogeneity (GI) index of ventilation and perfusion, dead space fraction, intrapulmonary shunt fraction, and V/Q matching was monitored using electrical impedance tomography (EIT). Additionally, respiratory mechanics, gas exchange, and hemodynamic parameters were recorded.A total of 24 patients with severe ARDS were enrolled, including 17 males and 7 females. The mean oxygenation index PaO2/FiO2 (P/F) at enrollment was 131.68 ± 33.11 mmHg. PP markedly improved static compliance (Cst),P/F and V/Q matching (p < 0.05), increased dorsal ventilation and perfusion, and decreased GI of ventilation and perfusion, as well as dead space ventilation (p < 0.05). Similarly, iNO significantly increased P/F and V/Q matching, while reducing perfusion GI, intrapulmonary shunt, and dead space ventilation (p < 0.05). Factorial analysis revealed that iNO was associated with a nonsignificant increase in the P/F ratio compared to the non-iNO group. (95 % CI: 6.037 to 84.927, p = 0.088). In contrast, PP significantly increased P/F compared to SP (95 % CI: 42.032 to 132.997, p < 0.001). The interaction effect between iNO and PP on P/F was not statistically significant (95 % CI: 63.099 to 65.544, p = 0.970). Regarding V/Q matching, iNO significantly improved outcomes compared to the non-iNO group (95 % CI: 1.902 to 15.363, p = 0.013), as did PP compared to SP (95 % CI: 1.255 to 14.717, p = 0.021). However, no significant interaction was observed between iNO and PP (95 % CI: 13.470 to 5.568, p = 0.412).In patients with moderate-to-severe ARDS, both iNO and PP are beneficial for improving V/Q matching and oxygenation. However, no combined effect of iNO and PP was observed.