Background: Activated vitamin D has anti-inflammatory properties. 25-Hydroxyvitamin D [25(OH)D] deficiency might contribute to subclinical interstitial lung disease (ILD). Objective: We examined associations between serum 25(OH)D concentrations and subclinical ILD among middle-aged to older adults who were free of cardiovascular disease at baseline. Methods: We studied 6302 Multi-Ethnic Study of Atherosclerosis (MESA) participants who had baseline serum 25(OH)D concentrations and computed tomography (CT) imaging spanning ≤ 10 y. Baseline cardiac CT scans (2000-2002) included partial lung fields. Some participants had follow-up cardiac CT scans at exams 2-5 and a full-lung CT scan at exam 5 (2010-2012), with a mean ± SD of 2.1 ± 1.0 scans. Subclinical ILD was defined quantitatively as high-attenuation areas (HAAs) between -600 and -250 Hounsfield units. We assessed associations of 25(OH)D with adjusted HAA volumes and HAA progression. We also examined associations between baseline 25(OH)D and the presence of interstitial lung abnormalities (ILAs) assessed qualitatively (yes or no) from full-lung CT scans at exam 5. Models were adjusted for sociodemographic characteristics, lifestyle factors (including smoking), and lung volumes. Results: The cohort's mean ± SD characteristics were 62.2 ± 10 y for age, 25.8 ± 10.9 ng/mL for 25(OH)D concentrations, and 28.3 ± 5.4 for body mass index (kg/m2); 53% were women, with 39% white, 27% black, 22% Hispanic, and 12% Chinese race/ethnicities. Thirty-three percent had replete (≥30 ng/mL), 35% intermediate (20 to <30 ng/mL), and 32% deficient (<20 ng/mL) 25(OH)D concentrations. Compared with those with replete concentrations, participants with 25(OH)D deficiency had greater adjusted HAA volume at baseline (2.7 cm3; 95% CI: 0.9, 4.5 cm3) and increased progression over a median of 4.3 y of follow-up (2.7 cm3; 95% CI: 0.9, 4.4 cm3) (P < 0.05). 25(OH)D deficiency was also associated with increased prevalence of ILAs 10 y later (OR: 1.5; 95% CI: 1.1, 2.2). Conclusions: Vitamin D deficiency is independently associated with subclinical ILD and its progression, based on both increased HAAs and ILAs, in a community-based population. Further studies are needed to examine whether vitamin D repletion can prevent ILD or slow its progression. The MESA cohort design is registered at www.clinicaltrials.gov as NCT00005487.