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BACKGROUND The mechanism of multifactorial spermatogenesis impairment is unclear. This study aims to investigate the reproductive toxicity of Pb in high-fat diet (HFD)-fed mice, and delineate the important role of gut microbiota. RESULTS Results showed that compared with normal diet (ND)-fed mice, Pb exposure caused more severe spermatogenesis impairment in HFD-fed mice, including decreased sperm count and motility, seminiferous tubule injury, serum and intratesticular testosterone decline, and downregulated expression level of spermatogenesis-related genes. Besides, 16S sequencing indicated that HFD-fed mice appeared more severer gut microbiota dysbiosis by Pb exposure than ND-fed mice. With fecal microbiota transplantation, the same trend of spermatogenesis impairment was occurred in recipient mice, which confirmed the important role of gut microbiota. Moreover, probiotics supplementation restored the gut microbial ecosystem, and thus improved spermatogenic function. CONCLUSION Our work suggested that population with HFD might face more reproductive health risks upon Pb exposure, and revealed an intimate linkage between microbiota dysbiosis and spermatogenesis impairment, accompanied with the potential usefulness of probiotics as prophylactic and therapeutic. This article is protected by copyright. All rights reserved.