Diminished transplacental glucose transport plays an important role in prenatal calorie restriction (CR) induced reduction in fetal growth. Fetal growth restriction (FGR) has an impact in shaping the adult phenotype with transgenerational implications. To understand the mechanisms underlying prenatal CR-induced transplacental glucose transport, we examined the epigenetic regulation of placental glucose transporter (Glut1 and Glut3) expression. We restricted calories by 50% in C57BL6 pregnant mice from gestational days 10 to 19 (CR; n=8) vs. controls (CON; n=8) and observed a 50% diminution in placental Glut3 expression (P<.05) with no effect on Glut1 expression by reverse transcription and quantitative real-time polymerase chain reaction (PCR). CR enhanced DNA methylation of a CpG island situated similar to 1000 bp upstream from the transcriptional start site of the glut3 gene, with no such effect on the glut1 gene as assessed by methylation-sensitive PCR and bisulfite sequencing. Chromatin immunoprecipitation (ChIP) assays demonstrated enhanced MeCP2 binding to the CpG island of the glut3 gene in response to CR vs. CON (P<.05). Sequential ChIP demonstrated that enhanced MeCP2 binding of the glut3-(m)CpG island enhanced histone deacetylase 2 recruitment (P<.05) but interfered with Sp1 binding (P<.001), although it did not affect Sp3 or Creb/pCreb interaction. We conclude that late-gestation CR enhanced DNA methylation of the placental glut3 gene. This epigenetic change augmented specific nuclear protein DNA complex formation that was associated with prenatal CR-induced reduction of placental glut3 expression and thereby transplacental glucose transport. This molecular complex provides novel targets for developing therapeutic interventions aimed at reversing FGR. (C) 2014 Elsevier Inc. All rights reserved.