The physiological and molecular mechanisms linking prenatal physical activity and improvements in maternal–fetal health are unknown. It is hypothesized that small extracellular vesicles (EVs, ~ 10–120 nm) are involved in tissue cross-talk during exercise. We aimed to characterize the circulating small EV profile of pregnant versus non-pregnant women after an acute bout of moderate-intensity exercise. Pregnant (N = 10) and non-pregnant control (N = 9) women performed a single session of moderate-intensity treadmill walking for 30 min. Plasma was collected immediately pre- and post-exercise, and small EVs were isolated by differential ultracentrifugation. EV presence was confirmed by western blotting for the small EV proteins TSG-101 and flottilin-1. Small EVs were quantified by size and concentration using nanoparticle tracking analysis and transmission electron microscopy. All EV fractions were positive for TSG-101 and flotillin-1, and negative for calnexin. Mean vesicle size at baseline and percent change in size post-exercise were not different between groups. At baseline, pregnant women had higher levels of small EVs compared to controls (1.83E+10 ± 1.25E+10 particles/mL vs. 8.11E+09 ± 4.04E+09 particles/mL, respectively; p = 0.032). Post-exercise, small EVs increased significantly in the circulation of pregnant compared to non-pregnant women after correcting for baseline values (64.7 ± 24.6% vs. − 23.3 ± 26.1%, respectively; F = 5.305, p = 0.035). Further research is needed to assess the functional roles of exercise-induced small EVs in pregnancy.