We evaluated the influence of different partial carbon dioxide pressure (PaCO2) levels on organ perfusion in patients with respiratory failure receiving pressure-support ventilation with veno-venous extracorporeal membrane oxygenation (V-V ECMO). In this twelve patients prospective study, ECMO gas-flow was decreased from baseline (PaCO2 < 40 mmHg) until PaCO2 increased by 5-10 mmHg (High-CO2 phase). Resistance indices of gut, spleen, and snuffbox artery, the peripheral perfusion index (PPI), and heart rate variability were measured at baseline and High-CO2 phase. When PaCO2 increased from 36 (36-37) mmHg at baseline to 42 (41-43) mmHg in the High-CO2 phase (p < 0.001), PPI decreased significantly (p = 0.026). The snuffbox artery (p = 0.022), superior mesenteric artery (p = 0.042), and spleen (p = 0.012) resistance indices increased significantly. The root mean square of successive differences (RMSSD) decreased from 19.5(18.1-22.7) to 15.9(14.4-18.6) ms (p = 0.034), and the ratio of low-frequency to high-frequency components(LF/HF) increased from 0.47 ± 0.23 to 0.70 ± 0.38 (p = 0.013). High PaCO2 might cause decreased peripheral tissue and visceral organ perfusion through autonomic nervous system in patients with respiratory failure undergoing PSV with V-V ECMO.