Background: Exposure to prenatal tobacco smoke (PTS) has been associated with a number of health outcomes in the offspring, including some childhood cancers. Lower levels of genomic DNA methylation have also been associated with several types of cancers. We investigated whether PTS was associated with global DNA methylation levels in the offspring. Methods: Our sample was drawn from a birth cohort of women born between 1959 and 1963 in New York City (n = 90). We measured methylation of repetitive elements (Sat2, Alu, LINE-1) from peripheral blood granulocytes. We combined prospectively collected data on PTS with adult epidemiologic data and blood samples collected in 2001 to 2007 (mean age, 43 years). We used linear regression to assess the association between PTS and repetitive element methylation. Results: Thirty-six percent of mothers smoked during pregnancy. We observed an inverse association between PTS and Sat2 methylation. This inverse association remained even after adjustment for potential mediators including child environmental tobacco smoke exposure, birth size, postnatal weight and height changes, and adult smoking status and alcohol intake (β=-0.22, 95% confidence interval =-0.40 to -0.03 for ever exposed to PTS vs. never exposed using models of log-transformed methylation levels). PTS exposure was not statistically significantly associated with LINE-1 or Alu methylation. Conclusions: PTS exposure, measured at the time of pregnancy and not retrospectively reported, was associated with a decrease in Sat2 methylation but not LINE-1 or Alu methylation. Impact: If replicated in larger studies, this study supports a persistent effect of PTS on DNA methylation levels, as measured by Sat2, in adulthood.