Background: The scientific literature does not supply enough information about the effects of postural changes on diffusing lung capacity for carbon monoxide (DLCO) in patients with chronic obstructive pulmonary disease (COPD), in particular regarding the prone position. Objectives: We evaluated posture-related changes in DLCO in healthy subjects and in COPD patients in order to especially assess how prone posture affects gas exchange. Methods: In this cross-sectional study, DLCO was measured in 10 healthy subjects and 30 COPD patients in standing, seated, supine and prone positions. Results: In healthy individuals, DLCO tended to improve from the upright to the supine position (21.42 ± 2.90 and 26.07 ± 5.11 ml/min/mm Hg, respectively); in the same group, changing the position from upright to prone also caused significant improvements in DLCO (absolute value, 21.42 ± 2.90 vs. 24.80 ± 4.39 ml/min/mm Hg, p < 0.05, or percent of predicted, 78.58 ± 11.12 vs. 91.44 ± 13.23, p < 0.05) and in DLCO proportional to alveolar volume (DLCO/VA; 4.52 ± 0.57 vs. 5.66 ± 1.48 ml/min/mm Hg/l, p < 0.05). No significant differences in DLCO have been observed in COPD patients from the standing to the prone position. Multivariate linear regression models showed that the posture-related changes in DLCO, DLCO expressed as percent of predicted and in DLCO/VA are directly correlated with the transition from upright/sitting to supine/prone. Conclusions: In healthy subjects, the effect of postural changes on DLCO could be explained by a more homogeneous perfusion, whereas the lack of variations in COPD patients could be attributed to the increased rigidity of lung capillaries, which could represent an early sensitive marker of damage of the alveolar capillary interface in these patients.