Oxidative stress is a biological imbalance in reactive oxygen species and antioxidants. Increased oxidative stress during pregnancy has been associated with adverse birth outcomes. Omega-3 fatty acid (n-3 FA) supplementation may decrease oxidative stress; however, this relationship is seldom examined during pregnancy. This study assessed the association between n-3 FA supplement use during pregnancy and urinary oxidative stress biomarker concentrations. Data came from The Infant Development and the Environment Study (TIDES), a prospective cohort study that recruited pregnant women in 4 US cities between 2010–2012. Third trimester n-3 FA intake was self-reported. Third trimester urinary 8-iso-prostaglandin F 2α (8-iso-PGF 2α ) was measured as an oxidative stress biomarker. Additionally, we measured the major metabolite of 8-iso-PGF 2α and Prostaglandin F 2α (PGF 2α ) and utilized the 8-iso-PGF 2α to PGF 2α ratio to calculate the change in 8-iso-PGF 2α reflecting oxidative stress versus inflammation. Adjusted linear models were used to determine associations with control for confounding. Of 725 women, 165 reported n-3 FA supplement use in the third trimester. In adjusted linear models, n-3 FA use was associated with 10.2% lower levels of 8-iso-PGF 2α (95% Confidence Interval [CI]: -19.6, 0.25) and 10.3% lower levels of the metabolite (95% CI: -17.1, -2.91). No associations were observed with PGF 2α . The lower levels of 8-iso-PGF 2α appeared to reflect a decrease in oxidative stress (percent change with supplement use: -18.7, 95% CI: -30.1, -5.32) rather than inflammation. Overall, third trimester n-3 FA intake was associated with lower concentrations of 8-iso-PGF 2α and its metabolite, suggesting a decrease in maternal oxidative stress during pregnancy.