In utero exposure to the phthalate ester plasticizer di-n-hexyl phthalate (DnHP) is known to affect the development of the male reproductive system and induce alterations in androgen-dependent tissues of male rat offspring. Male reproductive malformations produced by several phthalates have been causally linked to decreased testosterone production during the gestational period. This study was designed to evaluate the dose–response relationship for the effects of DnHP on the synthesis and production of testosterone in the fetal rat testis. Pregnant Sprague–Dawley rats were administered the vehicle (olive oil) and either DnHP (5 to 625 mg kg–1 per day) or diethylhexyl phthalate (DEHP) (50 or 625 mg kg–1 per day), by gavage, from gestation day (GD) 12 to19. Fetal testes were assessed on GD 19. DnHP reduced ex vivo testosterone production and down-regulated the expression of several genes required for cholesterol transport and steroid synthesis (i.e. SR-B1, StAR, P450scc, 3βHSD and P450c17). These inhibitions were dose dependent. A no-effect level was established at 5 mg kg–1 per day and a lowest-effect level at 20 mg kg–1 per day. mRNA levels of SR-B1, StAR, P450scc and 3βHSD were not similarly decreased in the adrenals. In conclusion, DnHP shares the same mode of action as DEHP in disrupting fetal testicular androgen synthesis. Alterations in testosterone production and in key steroidogenic gene expressions were apparent at lower doses than those causing postnatal reproductive malformations after gestational exposure during the critical period of male sexual differentiation. This suggests that they can be considered early biomarkers of DnHP-induced fetal testicular effects in rats. Copyright © 2013 John Wiley & Sons, Ltd.