We investigated the question of how serum zonulin levels change in intrahepatic cholestasis of pregnancy (ICP) and gestational diabetes mellitus (GDM) and, in the case of the coexistence of ICP and GDM, evaluated the eventual increase in zonulin plasmatic levels. Participants were enrolled for the study between 25 February 2021 and 20 August 2021. The prospective case-control study included: group 1 of 95 pregnant women diagnosed with ICP; group 2 of 110 pregnant women diagnosed with GDM; group 3 of 16 women diagnosed with both GDM and ICP; group 4 of 136 healthy pregnant women as the control group. The groups were compared in terms of age, body mass index (BMI), gravidity, parity, gestational week of delivery, plasma zonulin levels, delivery type, birth weight, first- and fifth-minute APGAR scores, newborn intensive care unit (NICU) admission, and meconium staining of amniotic fluid parameters. The results suggested that the plasma zonulin levels of ICP (group 1), GDM (group 2), and GDM with ICP (group 3) patients were higher than those of the healthy pregnant women of group 4 (p < 0.001). Among the patient groups, the highest median plasma zonulin levels were found in group 3 (110.33 ng/mL). Zonulin levels were also associated with the severity of ICP and adverse pregnancy outcomes. High serum zonulin levels were related to GDM, ICP, and adverse perinatal outcomes. The coexistence of GDM and ICP led to higher serum zonulin concentrations.