BACKGROUND: Gestational exposure to environmental chemicals (ECs) is associated with adverse, sex-specific offspring health effects of global concern. As the maternal steroid, cytokine and oxidative stress milieus can have critical effects on pregnancy outcomes and the programming of diseases in offspring, it is important to study the impact of real-life EC exposure, i.e., chronic low levels of mixtures of ECs on these milieus. Sheep exposed to biosolids, derived from human waste, is an impactful model representing the ECs humans are exposed to in real-life. Offspring of sheep grazed on biosolids-treated pasture are characterized by reproductive and metabolic disruptions. OBJECTIVE: To determine if biosolids exposure disrupts the maternal steroid, cytokine and oxidative stress milieus, in a fetal sex-specific manner. METHODS: Ewes were maintained before mating and through gestation on pastures fertilized with biosolids (BTP), or inorganic fertilizer (Control). From maternal plasma collected mid-gestation, 19 steroids, 14 cytokines, 6 oxidative stress markers were quantified. Unpaired t-test and ANOVA were used to test for differences between control and BTP groups (n = 15/group) and between groups based on fetal sex, respectively. Correlation between the different markers was assessed by Spearman correlation. RESULTS: Concentrations of the mineralocorticoids - deoxycorticosterone, corticosterone, the glucocorticoids - deoxycortisol, cortisol, cortisone, the sex steroids - androstenedione, dehydroepiandrosterone, 16-OH-progesterone and reactive oxygen metabolites were higher in the BTP ewes compared to Controls, while the proinflammatory cytokines IL-1ß and IL-17A and anti-inflammatory IL-36RA were decreased in the BTP group. BTP ewes with a female fetus had lower levels of IP-10. DISCUSSION: These findings suggest that pre-conceptional and gestational exposure to ECs in biosolids increases steroids, reactive oxygen metabolites and disrupts cytokines in maternal circulation, likely contributors to the aberrant phenotypic outcomes seen in offspring of BTP sheep - a translationally relevant precocial model.