It is unknown if high prolactin levels during pregnancy contribute to the development of gestational diabetes. We hypothesized that higher prolactin levels are associated with reduced glucose tolerance, as determined by higher 2-h glucose level from an oral glucose tolerance test in pregnancy. The 75-g oral glucose tolerance test was carried out at 28 weeks of gestation in 69 participants. A multiple regression analysis was used to determine the relationship between serum prolactin and 2-h glucose levels. Multivariable regression analysis showed an independent and significant relationship between third trimester prolactin and 2-h glucose levels post oral glucose tolerance test. Higher prolactin levels were associated with higher glucose levels independent of age, body mass index, gravidity and parity. Higher prolactin levels associated with reduced glucose tolerance in the third trimester of pregnancy suggests the possible independent role of prolactin in the pathogenesis of gestational diabetes.