The purpose of this study was to compare the prefrontal cortex (PFC) oxyhaemoglobin (HbO) levels observed during cycling in the upright and supine positions. Twelve healthy volunteers were randomly assigned to either the upright or supine position. After measuring maximal oxygen consumption (VOmax) with a cycle ergometer in the upright position, each subject exercised at an intensity corresponding to 50% of the VOmax for 20 min. HbO levels in the right PFC (R-PFC) and left PFC (L-PFC) were measured using near-infrared spectroscopy. The R-PFC HbO level measured within the 6-20-min window in the supine position was significantly higher than that in the upright position (0.072 ± 0.015 vs. 0.038 ± 0.013 mM·cm, respectively; p < 0.01). No significant effects were observed in the L-PFC. The laterality index for the supine position was significantly higher than that for the upright position (0.385 ± 0.181 vs. -0.272 ± 0.271, respectively; p < 0.05). Our results suggest that moderate-intensity cycling in the supine position increases R-PFC oxygenation.