Background: Some studies in animal models and humans suggest that exposure to lead is associated with hearing loss. Lead can reach the inner ear through the blood circulation; evidence suggests that lead could accumulate in the inner ear, causing inner ear damage. Aim: To evaluate prestin and otolin-1 protein levels and their relationship with an increased hearing threshold in participants exposed to lead. Methods: We conducted a cross-sectional study with 315 participants from Tlaxcala, Mexico. Blood lead levels (BPb) were evaluated by graphite furnace atomic absorption spectrometry. Serum prestin and otolin-1 were quantified using ELISA. Auditory function at frequencies of 0.125 to 8 kHz was evaluated in a soundproof chamber. Results: Participants were classified according to BPb: group I ( 0.05. Multiple linear regression models predict an average decrease of 0.17 to 0.26 ng/mL in prestin levels per decibel increase for the frequencies evaluated. Conclusions: Participants with high BPb showed an increase in hearing threshold, and prestin levels decreased proportionally to the hearing threshold increase. This is the first study to evaluate prestin as a potential biomarker for hearing damage, evaluated by audiometry, in participants with lead exposure.