Abstract: The aim of this investigation was to evaluate serotonin (5-HT) action on isolated human umbilical arteries (HUA) from normal and gestational diabetes mellitus (GDM) pregnancies. 5-HT caused HUA contraction in a concentration-dependent manner in both investigated groups but with lower efficacy in GDM. After endothelial denudation or in the presence of indomethacin (cyclooxygenase inhibitor), the 5-HT-evoked response was comparably augmented, but only in arteries from uncomplicated pregnancies. 5-HT contractions were unchanged by L-NOARG (NO-synthase inhibitor) or glibenclamide (KATP channel blocker) in both investigated groups. Whereas nifedipine (Ca2+ channel blocker) reduced the contractile effect of 5-HT and was more potent in GDM, ouabain (Na+/K+-ATPase inhibitor) caused the contraction of HUAprior to 5-HT addition in both groups, but with a significantly reduced effect in GDM. In vascular rings from GDM, methiothepin (a 5-HT1/5-HT2 receptor antagonist) significantly reduced 5-HT-induced contraction to a similar extent as compared to uncomplicated pregnancies. Ketanserin (a 5-HT2A receptor antagonist) produced a concentration-dependent inhibition of the 5-HT effect in GDM. In conclusion, in normal pregnancies, 5-HT produced a concentration- and endothelium-dependent contraction of HUA, most probably via endothelial prostacyclin. In contrast, the contractile effect of 5-HT in GDM was reduced with apparent endothelial dysfunction. In both normal and diabetic pregnancies, voltage-gated Ca2+ channels and Na+/K+-ATPase contribute to the 5-HT-evoked contraction, as well to the regulation of basal vascular tone, but those actions were notably impaired in GDM. In uncomplicated and diabetic pregnancies, the transduction mechanism of 5-HT involves activation of mixed population of 5-HT1 and 5-HT2A receptors in the HUA.