To assess if anti-mullerian hormone (AMH) concentrations decrease during the third trimester of pregnancy and puerperium and whether this is correlated to gestational diabetes mellitus (GDM). AMH serum concentrations were determined by ELISA, with lowest detection limit of 0.08 ng/ml, during the third trimester of pregnancy and puerperium in 34 patients with GDM and in 32 healthy control pregnant women. Three blood samples were collected at 28-32, 34-36 weeks' gestation and 40 days after delivery, respectively. No differences in AMH concentration between GDM and healthy pregnant women were found at any follow-up time. On the contrary, significant differences in delta AMH values between the first and the second sample (p < 0.0001), the second and the third sample (p < 0.0001), and the first and the third sample (p = 0.004) were found in both groups. The multivariate analysis showed that maternal age was not correlated to delta AMH variations. The analysis of AMH concentrations did not show a significant relationship with body mass index-variation, newborn and placental weight, and GDM, while only maternal age was significantly correlated with AMH concentrations in all blood samples. A significant decrease in AMH values was observed during the third trimester of pregnancy and this was independent on maternal age. GDM does not seem to influence the AMH concentrations during pregnancy and after delivery.