This study was designed to investigate the impact of lead (Pb 2+ ) on the auditory system and its molecular mechanisms. Pb(AC) 2 was administrated to male SD rats aged 21–22 d for 8 weeks at a dose of 300ppm. Male guinea pigs were also administrated with 50mg/kg Pb(AC) 2 two times a week for 8 weeks. The auditory nerve–brainstem evoked responses (ABR) was recorded and the morphological changes of the outer hair cells (OHCs) were observed with Phallodin-FITC staining. In addition, the integrity of the blood–labyrinth barrier was observed by TEM and the expression of tight junction proteins (TJPs) in the cochlear stria vascularis was determined by immunofluorescence. Our results showed that Pb 2+ exposure resulted in increased ABR threshold in both rats and guinea pigs. Abnormal shapes and loss of OHCs were found in the cochlear basilar membrane following the Pb 2+ exposure. TEM study showed that the tight junctions between the endothelial cells and the border cells were lost and disrupted. Down-regulation of the occludin, ZO-1 and claudin-5 in the stria vascularis suggested that the increased permeability of the blood–labyrinth barrier may attribute to the Pb 2+ -induced decrease of TJPs’ expression. Additionally, Fe 2+ supplement partly reversed the Pb 2+ -induced hearing loss and down-regulation of TJPs. Taken together, these data indicate that the disruption of blood–labyrinth barrier by down-regulating the expression of TJPs plays a role in the Pb 2+ -induced hearing loss, and Fe 2+ supplement protects the auditory system against Pb 2+ -induced toxicity and may have significant clinical implications.