Di-2-ethylhexyl phthalate (DEHP) is a common plasticizer with a deleterious impact on testicular functionality and male fertility. Growing evidence implicates ferroptosis as one of the plausible mechanisms for DEHP-induced testicular injury. Sulforaphane (SFN) is a natural isothiocyanate displaying beneficial effects on testicular injury in several animal models. Herein, we explored the potential protective effect of SFN on testicular ferroptosis and toxicity evoked by DEHP. Adult male Wistar rats were equally distributed into three groups (n = 6/group): (i) CON group
(ii) DEHP group, received DEHP (2 g/kg PO) for 4 weeks
and (iii) DEHP + SFN group, received SFN (10 mg/kg, PO) 1 week prior to DEHP then concurrently with DEHP for further 4 weeks. Compared to CON group, exposure to DEHP caused testicular atrophy, deteriorated testicular architecture, testicular fibrosis, reduced sperm count and motility, higher sperm deformity, and declined serum testosterone level. All these abnormalities were ameliorated by SFN preconditioning. Additionally, pretreatment with SFN reversed the increased aromatase level and upregulated the steroidogenic markers in testes of DEHP-exposed rats. SFN pretreatment also counteracted DEHP-induced oxidative stress and boosted the total antioxidant capacity in testicular tissue via activation of the nuclear factor erythroid 2-related factor 2 (NRF-2) and its downstream target, hemeoxygenase-1 (HO-1). Moreover, SFN preconditioning mitigated DEHP-induced ferroptosis through up-surging SLC7A11, GPX-4, and GSH, while suppressing iron overload and ACSL4-induced lipid peroxidation in testicular tissue of rats. These findings may nominate SFN as a promising protective intervention to alleviate testicular ferroptosis associated with DEHP exposure through activation of NRF-2/SLC7A11/GPX-4 trajectory.