Although both insulin and estrogen receptor alpha (ER alpha) are known to exert inhibitory effects on testicular steroidogenesis, it remains unknown whether these pathways regulate testosterone (T) production under certain pathological conditions [e.g., type 2 diabetes mellitus (T2DM)] in a coordinated manner. Here, we found that the expression of forkhead box protein A3 (Foxa3), an essential transcriptional regulator engaged in adipogenesis and energy metabolism, was significantly down-regulated in the Leydig cells (LCs) from T-deficient T2DM mice. Functionally, upon hCG stimulation, Foxa3 recruits to the Esr1 promoter and suppresses the transactivation of Esr1 gene. Disruption of this recruitment by T2DM-elicited hyperinsulinemia led to abnormal activation of ER alpha pathway, inhibited steroidogenic enzyme genes expression, and thus caused inadequate T production. Therapeutically, insulin-impaired and Foxa3 ablation-compromised steroidogenesis were effectively rescued by a pharmacological inhibitor of the ER alpha pathway. These findings reveal an obligatory coregulatory role of Foxa3 in the regulation of ER alpha expression and of the Foxa3/ER alpha cascade, at least in part, in the pathogenesis of androgen deficiency caused by T2DM. (C) 2017 Elsevier Inc. All rights reserved.