This study was designed to assess glucose homeostasis in pregnant women in their third trimester of gestation in response to exercise. Specifically, this study was designed to (1) compare the extend and rate at which blood glucose levels decrease in pregnant (22 to 33 weeks of gestation) versus that which occurs in nonpregnant women; and (2) determine the pattern of changes of the substrates (glucose, lactate, beta-hydroxybutyrate, and free fatty acids, and hormones (insulin), that contribute to the glucose homeostasis of pregnant (N = 10), and nonpregnant (N = 10) women in response to 1-hour prolonged moderate intensity exercise (at 55% of their VO2max). Each subject was tested for the determination of their maximal oxygen consumption (VO2max) and, based on their VO2max, they performed 60 minutes of prolonged moderate intensity exercise. Blood was collected before, during, and after the exercise bout. The results indicated that blood glucose levels of pregnant women decrease at a faster rate and to a significantly lower level post exercise (P < .05). Insulin levels of pregnant women also decreased to a significantly lower level post exercise, and lactate levels were maintained at a lower level 15 minutes after exercise. beta-hydroxybutyrate level was not different between the two groups, but demonstrated a different pattern of changes during exercise (P < .05). Furthermore, the results suggest that blood glucose levels of the late pregnant women decrease lower than those of nonpregnant women; also, there are differences in the rate and kinetics of blood glucose between pregnant and nonpregnant women. The results also indicate significant differences in the level of circulating substrates and hormones between pregnant and nonpregnant women in response to exercise.