Background: Approximately 40% of women are prescribed an antibiotic during pregnancy. Though completed course of therapy is necessary for treatment of infections, antibiotic use has been associated with several adverse birth outcomes. Biological changes in the prenatal environment affected by antibiotic use are poorly understood. Specifically, examining maternal epigenetic patterns associated with antibiotic use can identify CpG sites that may provide insight into biological pathways associated with adverse birth outcomes.Objectives: To evaluate the association of antibiotic use, abstracted from medical records as participants who had ever been prescribed an antibiotic during pregnancy, and maternal peripheral blood DNA methylation patterns during pregnancy in the MADRES (Maternal and Developmental Risks from Environmental and Social Stressors) study, a pregnancy cohort of predominantly lower income Hispanic women in Los Angeles. A paired, longitudinal analysis was used to evaluate changes in methylation between the first and third trimester in women who had ever been prescribed an antibiotic during pregnancy. Exploration of discrete effects at the first and third trimester was also undertaken, as contraindication of antibiotic differs across trimesters of pregnancy.Methods: Antibiotic use, indicated by prescription of antibiotic, and its association with differentially methylated positions (DMPs) on the Illumina EPIC array were analyzed using linear mixed and linear regression models. Estimated blood cell type fractions and surrogate variables were used to control for both measured and unmeasured confounding variables in statistical analysis.Results: In the third trimester, 15 DMPs were identified (PFDR<0.05) in relation to antibiotic use during pregnancy. Antibiotic use during pregnancy was not associated with first to third trimester methylation changes for any loci on the EPIC array.Conclusion: In the MADRES cohort, antibiotic use during pregnancy was not associated with changes in methylation during pregnancy, but was associated with differential methylation at 15 CpGs in third trimester peripheral blood samples.