Perturbation of epigenetic regulation is a well-established mechanism for cancer but its role for lead (Pb)-associated toxicity has not been adequately investigated. We aimed to investigate whether occupational Pb exposure is associated with micronuclei (MN) frequency and to further explored the mediating roles of epigenetic gene regulation. All the Pb-exposed workers recruited from a Chinese acid battery factory, blood lead levels (BLLs) and MN frequency in lymphocytes were measured. In addition, methylation levels of seven genes (Line-1, RASSF1A, RUNX3, p16, CYP26C1, hMLH1, p15) were examined among 230 workers. Robust Poisson regression model was used to investigate the association between BLLs and MN frequency. Mediation analysis was used to explore the mediating role of specific DNA methylation. Among total 677 participants, 71% were male, median BLLs was 229.1 μg/L (P25 = 155.5, P75 = 319.3; ranged from 8.9 to 647.7, μg/L), mean MN frequency was 2.5 ‰ (SD = 1.8 ‰; ranged from 0 ‰ to 9 ‰). Results from base model, adjusted for age, sex, and body mass index, showed that MN frequency would increase 1.38 (95%CI: 1.34, 1.43) per 100 μg/L increment in BLLs. Using categorized exposure variable analyses, a BLLs dose–response increase in MN frequency was observed: 2.74 (2.13, 3.51), 3.43 (2.73, 4.32), 4.41 (3.89, 5.01) to 6.86 (6.02, 7.81). Mediation analysis indicated that Line-1 methylation significantly mediated 3.6% of the association of BLLs with MN frequency. Occupational Pb exposure induces MN frequency in a dose–response relationship. Part of this association was mediated by Line-1 promotor methylation.