Introduction: Phthalates are widely found in consumer products and phthalate metabolites are regularly detected in human urine. Phthalates have anti-androgenic activity and have been associated with shorter anogenital distance (AGD) in male infants. AGD is sexually dimorphic (longer in boys than girls) and an indicator of fetal androgen exposure. Our study assessed AGD in relation to prenatal exposure to phthalates. Methods: Phthalate metabolites were quantified in a pool of 5 spot urines collected across pregnancy from 149 participants in a prospective cohort in the Midwestern U.S. recruited from 2010 to 2012. We focused on the sum of di-2-ethylhexyl phthalate (DEHP) metabolites and also created a sum of the anti-androgenic phthalates, adjusted for specific gravity. After birth, trained study staff measured anoscrotal distance (AGD-AS) and anopenile distance (AGD-AP) in males and anofourchette distance (AGD-AF) and anoclitoral distance (AGD-AC) in females. We used multivariable linear regression adjusted for infant's age at exam, weight-for-length z-score, gestational age at birth; maternal race/ethnicity, pregnancy smoking, age, income, and pre-pregnancy body mass index. Results: Urinary concentrations of phthalate metabolites were similar to levels in other U.S. populations. Each µmol/L increase in the sum of DEHP metabolites was significantly associated with AGD-AS in male infants (β: -6.7 mm, 95% confidence intervals (CI): -12.4, -1.1). There was a suggestion of an inverse association with each µmol/L increase in anti-androgenic phthalates for both AGD-AS and AGD-AP (β: -0.7 mm, 95% CI:-2.8, 1.4 and β: -1.1 mm, 95% CI:-4.1, 1.8, respectively). For females, a non-significant decrease in AGD-AF was observed with DEHP and anti-androgenic phthalates (β: -0.7 mm, 95% CI:-1.8, 0.5 and β: -0.6 mm, 95% CI:-1.6, 0.5, respectively). Conclusions: Our findings support the hypothesis that phthalates may affect reproductive development in infants, particularly males.