Purpose: Chronic obstructive pulmonary disease (COPD) is an irreversible lung disease characterized by small-airway obstruction and alveolar-airspace destruction. Hyperpolarized 129Xe diffusion MRI of lung is a promising biomarker for assessing airspace enlargement, but has yet to be validated by direct comparison to lung histology. Here we have compared diffusion measurements of hyperpolarized (HP) 129Xe in explanted lungs to regionally matched morphological measures of airspace size. Methods: Explanted lungs from five COPD patients and two idiopathic pulmonary fibrosis (IPF) patients were imaged using MRI with hyperpolarized 129Xe using a two-b-value gradient-echo diffusion sequence, and 34 histological samples were taken from these lungs for quantitative histology. Mean-linear-intercept (Lm) was compared with spatially matched measures of apparent diffusion coefficient (ADC) from 129Xe MRI. Results: The mean ADC from COPD lung samples was 0.071 ± 0.011 cm2/s, and for IPF lungs was 0.033 ± 0.001 cm2/s (P < 10−15 between groups). The mean Lm in COPD samples was 0.076 ± 0.027 cm and 0.041 ± 0.004 cm in IPF (P = 2.7 × 10−7 between groups). The Pearson-correlation between ADC and Lm measurements was r = 0.59. Conclusions: Diffusion MRI of HP 129Xe quantifies regional airspace enlargement in COPD. 129Xe ADC showed much less overlap between groups than quantitative histology, consistent with our past experience with 3He diffusion MRI in COPD. Magn Reson Med 77:265–272, 2017.