Perfluorohexane sulfonic acid (PFHxS) is extensively used in waterproof coatings and fire-fighting foams, and several studies have found it to be a potential health hazard, but there is still unknown about its effects on spermatogenesis. Our results showed that PFHxS-treated mice have significant reproductive toxicity, including a decrease in sperm count and motility, and the levels of sex hormones (P <
0.05). Concurrently, structural abnormalities are observed in sperm, affecting ≈60-75% of those in the PFHxS-treated group. Additionally, it is found that the structure of the blood-testis barrier (BTB) is damaged after PFHxS treatment, leading to higher expression levels of inflammatory cytokines in the microenvironment for spermatogenesis. Moreover, the expression of proteins associated with mitochondrial biogenesis, including PTEN-induced kinase 1 (PINK1) and NADPH oxidase 4 (NOX4), is dysregulated in the testes after PFHxS treatment. Based on metabolome data, the differential metabolite 3-hydroxybutanoic acid is identified in the PFHxS-treated group, which can regulate the histone Kac levels, especially H3K4ac and H3K9ac. In summary, the results of this study suggest that in the testes of PFHxS-treated mice, inflammatory factors disrupt the mitochondrial function and metabolic profiles and hinder the progress of gene transcription through histone Kac, ultimately causing sperm dysfunction.