Failure of the testes to descend into the scrotum (cryptorchidism) is one of the most common birth defects in humans. In utero exposure to estrogens, such as 17β-estradiol (E2) or the synthetic estrogen diethylstilbestrol (DES), down-regulates insulin-like 3 (Insl3) expression in embryonic Leydig cells, which in turn results in cryptorchidism in mice. To identify the molecular mechanism whereby xenoestrogens block Insl3 gene transcription, we performed a microarray analysis of wild-type or estrogen receptor (ER) α-mutant testes exposed in utero to pharmacological doses of E2 or DES. Six and 31 genes were respectively down-regulated and up-regulated by estrogen exposure (≥4-fold). All six genes down-regulated by estrogen exposure, including Insl3 and the steroidogenic genes steroidogenic acute regulatory protein and cytochrome P450 17α-hydroxylase/17,20-lyase, were done so by an ERα-dependent mechanism. In contrast, up-regulation was mediated either by ERα for 12 genes or by an independent mechanism for the 19 remaining genes. Finally, we show that Insl3 gene expression and testicular descent were not affected by in utero exposure to E2 or DES in ERα mutant mice, whereas absence of ERβ did not influence the effect of these estrogens. Collectively, these data demonstrate that xenoestrogens inhibit the endocrine functions of fetal Leydig cells through an ERα-dependent mechanism.