Currently, no clinical studies have compared the inspiratory and expiratory volumes of unilateral lung or of each lobe among supine, standing, and sitting positions. In this prospective study, 100 asymptomatic volunteers underwent both low-radiation-dose conventional (supine position, with arms raised) and upright computed tomography (CT) (standing and sitting positions, with arms down) during inspiration and expiration breath-holds and pulmonary function test (PFT) on the same day. We compared the inspiratory/expiratory lung/lobe volumes on CT in the three positions. The inspiratory and expiratory bilateral upper and lower lobe and lung volumes were significantly higher in the standing/sitting positions than in the supine position (5.3–14.7% increases, all P < 0.001). However, the inspiratory right middle lobe volume remained similar in the three positions (all P > 0.15); the expiratory right middle lobe volume was significantly lower in the standing/sitting positions (16.3/14.1% decrease) than in the supine position (both P < 0.0001). The Pearson’s correlation coefficients (r) used to compare the total lung volumes on inspiratory CT in the supine/standing/sitting positions and the total lung capacity on PFT were 0.83/0.93/0.95, respectively. The r values comparing the total lung volumes on expiratory CT in the supine/standing/sitting positions and the functional residual capacity on PFT were 0.83/0.85/0.82, respectively. The r values comparing the total lung volume changes from expiration to inspiration on CT in the supine/standing/sitting positions and the inspiratory capacity on PFT were 0.53/0.62/0.65, respectively. The study results could impact preoperative CT volumetry of the lung in lung cancer patients (before lobectomy) for the prediction of postoperative residual pulmonary function, and could be used as the basis for elucidating undetermined pathological mechanisms. Furthermore, in addition to morphological evaluation of the chest, inspiratory and expiratory upright CT may be used as an alternative tool to predict lung volumes such as total lung capacity, functional residual capacity, and inspiratory capacity in situation in which PFT cannot be performed such as during an infectious disease pandemic, with relatively more accurate predictability compared with conventional supine CT.