•HUVEC exposed to a GDM milieu in vivo has reduced migratory capacity.•Dysregulated expression of TGF-β receptors is detected.•Human fetal endothelium of GDM women responds abnormally to TGF-β1.•Several genes involved in development, cell movement and migration were downregulated.•Altered transcriptome may affect cardiac morphogenesis and placental development. Enhanced biosynthesis of several cytokines, such as, transforming growth factor-β1 (TGF-β1), is detected in gestational diabetes mellitus (GDM). In this study, we addressed the question of whether the exposure to the abnormal milieu of GDM in vivo affects gene expression pattern of human umbilical vein endothelial cells (HUVEC) in response to TGF-β1. We found that HUVEC isolated from GDM (dHUVEC) had reduced migratory capacity versus those of healthy women (nHUVEC) and this quiescent phenotype was associated with higher expression levels of the TGF-βtype I receptor ALK5 and a slight increase in the endogenous production of TGF-β1 (mainly in its latent form). Moreover, we performed transcriptome analysis, using microarray technology, of dHUVEC versus nHUVEC, after 3h treatment with exogenous TGF-β1 (10ng/ml). The treatment of dHUVEC with TGF-β1 caused downregulation of the transcription of multiple genes involved in development, cell movement and migration of cells versus TGF-β1-treated nHUVEC. These changes in transcriptome profile might contribute to GDM-dependent alterations in cardiac morphogenesis and placental development.