While numerous studies have shown that fluoride or arsenic exposure may damage the reproductive system, there are few reports of co-exposure to fluoride and arsenic. In addition, the literature on autophagy and intestinal flora composition in reproductive toxicity studies of co-exposure to fluoride and arsenic is insufficient. In this study, we developed a rat model of fluoride and arsenic exposure via drinking water from pre-pregnancy to 90 days postnatal. Sprague-Dawley rats were randomly divided into sterile water control group, fluoride group (100 mg/L NaF), arsenic group (50 mg/L NaAsO) and combined exposure group (100 mg/L NaF+50 mg/L NaAsO). Our results showed that fluoride and arsenic exposure caused a reduction in testicular weight and significant pathological damage to tissue. We found that the levels of follicle-stimulating hormone, luteinizing hormone, and testosterone were reduced to varying degrees. Meanwhile experiments showed that fluoride and arsenic exposure can modulate autophagic flux, causing increased levels of Beclin1 and LC3 expression and decreased p62 expression. Analogously, by performing 16S sequencing of rat feces, we found 24 enterobacterial genera that differed significantly among the groups. Furthermore, the flora associated with testicular injury were identified by correlation analysis of hormonal indices and autophagy alterations with intestinal flora composition at the genus level, respectively. In summary, our study shows that fluoride and arsenic co-exposure alters autophagic flux in the testis, causes testicular injury, and reveals an association between altered intestinal flora composition and testicular injury.