Objective: Determine the effect of physical activity on vitamin D (vitD) status in pregnant women. Design/Methods: Women who presented at 10-14 weeks' gestation participated in vitD supplementation RCT, conducted between November 2012 and June 2016, were randomized into one of two treatment groups: 400 or 4400 IU/day of vitD3. VitD status, as defined by total circulating 25(OH)D concentration, was measured monthly. Physical activity was measured monthly using the Paffenbarger Questionnaire, and responses were categorized as vigorously exercising for ≥ 2.5 h/week during each trimester. Sunlight exposure was measured using skin spectrophotometry (SmartProbe 400). Linear regression and mixed models explored the association of maternal 25(OH)D concentration, sunlight exposure, physical activity, race, treatment group, body mass index, perceived stress score, and visit using SAS software (version 9.4). Results: 211 pregnant women had physical activity information available. At baseline, vigorously exercising ≥ 2.5 h/week was associated with being older (p=0.0004), white/Caucasian (p=0.011), privately insured (p=0.018), and purposefully pregnant (p<0.031). Baseline 25(OH)D did not vary by treatment or exercise groups. In bivariate analysis, at second trimester, vigorously exercising ≥ 2.5 h/week was associated with being white/Caucasian, privately insured, purposefully pregnant, married and with higher 25(OH)D concentration. By third trimester, vigorous exercise was associated with being privately insured, married, gravidity, sunlight exposure and Hispanic. The effect of increased exercise on 25(OH)D concentration was seen only in the 400 IU group at third trimester. In a mixed model controlling for multiple potential confounders, maternal vitD status and exercise were positively associated (p=0.018). Conclusion: Independent of sunlight exposure, pregnant women who vigorously exercised ≥ 2.5 h/week had higher 25(OH)D concentrations than those who did not. Supplementation may have overshadowed the exercise effect; the positive correlation was amplified when looking at the 400 IU group alone, and may suggest that exercise was more critical for those lacking the higher supplementation dose.