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Particulate matter with an aerodynamic diameter of 2.5 μm or less (PM_2.5) is a ubiquitous air pollutant that is increasingly threatening the health of adults and children worldwide. One health impact of elevated PM_2.5 exposure is alterations in telomere length (TL)—protective caps on chromosome ends that shorten with each cell division. Few analyses involve prenatal PM_2.5 exposure, and paired maternal and cord TL measurements. Here, we analyzed the association between average and trimester-specific prenatal PM_2.5 exposure, and maternal and newborn relative leukocyte TL measured at birth among 193 mothers and their newborns enrolled in a New-York-City-based birth cohort. Results indicated an overall negative relationship between prenatal PM_2.5 and maternal TL at delivery, with a significant association observed in the second trimester (β = −0.039, 95% CI: −0.074, −0.003). PM_2.5 exposure in trimester two was also inversely related to cord TL; however, this result did not reach statistical significance (β = −0.037, 95% CI: −0.114, 0.039), and no clear pattern emerged between PM_2.5 and cord TL across the different exposure periods. Our analysis contributes to a limited body of research on ambient air pollution and human telomeres, and emphasizes the need for continued investigation into how PM_2.5 exposure during pregnancy influences maternal and newborn health.