Abstract Metastable epialleles (MEs) are genomic regions that are stochastically methylated prior to germ layer specification and exhibit high interindividual but low intra-individual variability across tissues. ME methylation is vulnerable to environmental stressors, including diet. Tobacco smoke (TS) exposure during pregnancy is associated with adverse impacts on fetal health and maternal micronutrient levels as well as altered methylation. Our objective was to determine if maternal smoke exposure impacts methylation at MEs. Consistent with prior studies, we observed reductions in one-carbon pathway micronutrients with gestational TS exposure, including maternal folate (P = 0.02) and vitamins B6 (P = 0.05) and B12 (P = 0.007). We examined putative MEs BOLA3, PAX8, and ZFYVE28 in cord blood specimens from 85 Newborn Epigenetics STudy participants. Gestational TS exposure was associated with elevated DNA methylation at PAX8 (+5.22% average methylation; 95% CI: 0.33% to 10.10%; P = 0.037). In human conceptal kidney tissues, higher PAX8 transcription was associated with lower methylation (Rs = 0.55; P = 0.07), suggesting that the methylation levels established at MEs, and their environmentally induced perturbation, may have meaningful, tissue-specific functional consequences. This may be particularly important because PAX8 is implicated in several cancers, including pediatric kidney cancer. Our data are the first to indicate vulnerability of human ME methylation establishment to TS exposure, with a general trend of increasing levels of methylation at these loci. Further investigation is needed to determine how TS exposure-mediated changes in DNA methylation at MEs, and consequent expression levels, might affect smoking-related disease risk.