Leydig cell tumours (LCTs) are frequently observed during rodent carcinogenicity studies, however, the significance of this effect to humans remains a matter of debate. Many chemicals that produce LCTs also induce hepatic cytochromes P450 (CYPs), but it is unknown whether these two phenomena are causally related. Our aim was to investigate the existence of a liver-testis axis wherein microsomal enzyme inducers enhance testosterone metabolic clearance, resulting in a drop in circulating hormone levels and a consequent hypertrophic response from the hypothalamic-pituitary-testis axis. Lansoprazole was selected as the model compound as it induces hepatic CYPs and produces LCTs in rats. Male Sprague-Dawley rats were dosed with lansoprazole (150 mg/kg/day) or vehicle for 14 days. Lansoprazole treatment produced effects on the liver consistent with an enhanced metabolic capacity, including significant increases in relative liver weights, total microsomal CYP content, individual CYP protein levels, and enhanced CYP-dependent testosterone metabolism in vitro. Following intravenous administration of [14C]testosterone, lansoprazole-treated rats exhibited a significantly smaller area under the curve and significantly higher plasma clearance. Significant reductions in plasma and testicular testosterone levels were observed, confirming the ability of this compound to perturb androgen homeostasis. No significant changes in plasma LH, FSH, or prolactin levels were detected under our experimental conditions. Lansoprazole treatment exerted no marked effects on testicular testosterone metabolism. In summary, lansoprazole treatment induced hepatic CYP-dependent testosterone metabolism in vitro and enhanced plasma clearance of radiolabelled testosterone in vivo. These effects may contribute to depletion of circulating testosterone levels and hence play a role in the mode of LCT induction in lansoprazole-treated rats.