Immersion in high concentrations of CO2 dissolved in freshwater (CO2-water) might induce peripheral vasodilatation in humans. In this study, we investigated whether such immersion could affect the autonomic nervous system in humans using spectral analysis of heart rate variability. Ten healthy men participated in this study. Tympanic temperature, cutaneous blood flow and electrocardiogram (ECG) were measured continuously during 20 min of immersion in CO2-water. The ECG was analyzed by spectral analysis of R-R intervals using the maximal entropy method. The decrease in tympanic temperature was significantly greater in CO2-water immersion than in freshwater immersion. Cutaneous blood flow at the immersed site was significantly increased with CO2-water immersion compared to freshwater. The high frequency component (HF: 0.15-0.40 Hz) was significantly higher in CO2-water immersion than in freshwater immersion, but the low frequency (LF: 0.04-0.15 Hz) /high frequency ratio (LF/HF ratio) was significantly lower in CO2-water immersion than in freshwater immersion. The present study contributes evidence supporting the hypothesis that CO2-water immersion activates parasympathetic nerve activity in humans.