Zearalenone (ZEA), a fungal mycotoxin, is present in a wide range of human foods. By virtual screening, we have identified that ZEA is a potential endocrine disruptor of Leydig cells. The effect of ZEA on Leydig cell development is still unclear. The objective of the present study was to explore whether ZEA affected Leydig cell developmental process and to clarify the underlying mechanism. Adult male Sprague-Dawley rats (60 days old) were randomly divided into three groups and these rats received a single intraperitoneal injection of 75 mg/kg ethane dimethane sulfonate (EDS) to eliminate all Leydig cells. Seven days after EDS treatment, rats intratesticularly received normal saline (control) or 150 or 300 ng/testis/day ZEA for 21 days. Immature Leydig cells isolated from 35-day-old rats were treated with ZEA (0.05-50 μM) for 24 h in vitro. In vivo ZEA exposure lowered serum testosterone levels, reduced Leydig cell number, and decreased Leydig cell-specific gene or protein expression levels possibly via downregulating the steroidogenic factor 1 (Nr5a1) expression. ZEA in vitro inhibited androgen production and steroidogenic enzyme activities in immature Leydig cells by downregulating expression levels of cholesterol side cleavage enzyme (Cyp11a1), 3β-hydroxysteroid dehydrogenase 1 (Hsd3b1), and steroid 5α-reductase 1 (Srd5a1) at a concentration as low as 50 nM. In conclusion, ZEA exposure disrupts Leydig cell development and steroidogenesis possibly via downregulating Nr5a1.