Urocortin is produced by the placenta throughout pregnancy but its regulation remains unknown. The effect of hypoxia on placental urocortin production is not known. The aim of this study was to determine the effect of in vitro hypoxia on human trophoblastic urocortin production. Placental explants and primary cultures were incubated in anaerobe hypoxic bags for 24 h in a humidified incubator. Urocortin peptide secretion and mRNA (messenger RNA) production was determined by enzyme-linked immunosorbent assay and reverse transcription-polymerase chain reaction, respectively. Morphological and functional integrity was verified by immunohistochemical analysis of urocortin expression. Vascular endothelial growth factor expression was used to verify the generation of cellular hypoxia in our in vitro system. Hypoxia did not affect urocortin secretion or mRNA expression in explant and single-cell cultures. Production was greater from first trimester than term explants and from single-cell primary cultures more than from explant cultures. Hypoxia does not influence human placental urocortin secretion or mRNA expression in vitro.