Background: Little is known about the possible deleterious effects of phthalate exposure on endogenous sex steroid levels in children. Objective: Our objective was to investigate whether urinary phthalate metabolite levels are associated with circulating adrenal androgen levels and age at puberty. Methods: This was a longitudinal study of 168 healthy children (84 girls) examined every 6 months for 5 years. Serum levels of dehydroepiandrostenedione sulfate (DHEAS), Δ4-androstenedione, testosterone, and urinary morning excretion of 14 phthalate metabolites, corresponding to 7 different phthalate diesters were determined. A variation in urinary excretion of phthalates was evident in each child, which made a mean of repetitive samples more representative for long-term excretion than a single determination. Results: We found that girls with excretion of monobutyl phthalate isomers (MBP) and di(2-ethylhexyl) phthalate metabolites above the geometric group mean (795 and 730 ng/kg, respectively) had lower levels of DHEAS and Δ4-androstenedione, although statistically significant only at 13 years of age. In boys, we found that excretion of monobenzyl phthalate above the geometric group mean (346 ng/kg) was associated with lower levels of DHEAS at 11 years of age but higher levels of testosterone at 13 years of age. The same trend was observed for MBP excretion, albeit not statistically significant. A lower age at pubarche was observed in boys with excretion of MBP above the geometric group mean (11.0 vs 12.3 years, P = 0.005). Conclusion: Our data indicate that exposure to dibutyl phthalate isomers (DBP) (in girls) and butylbenzyl phthalate (in boys) are negatively associated with adrenal androgen levels and in boys positively associated with testosterone level at 13 years of age. High exposure to DBP was associated with earlier age at pubarche in boys. In girls, no associations between phthalate exposure and age at pubertal milestones were observed.