Effects of the diet-induced gut microbiota dysbiosis reach far beyond the gut. We aim to uncover the direct evidence involving the gut-testis axis in the aetiology of impaired spermatogenesis.An excessive-energy diet-induced metabolic syndrome (MetS) sheep model was established. The testicular samples, host metabolomes and gut microbiome were analysed. Faecal microbiota transplantation (FMT) confirmed the linkage between gut microbiota and spermatogenesis.We demonstrated that the number of arrested spermatogonia was markedly elevated by using 10× single-cell RNA-seq in the MetS model. Furthermore, through using metabolomics profiling and 16S rDNA-seq, we discovered that the absorption of vitamin A in the gut was abolished due to a notable reduction of bile acid levels, which was significantly associated with reduced abundance of Ruminococcaceae_NK4A214_group. Notably, the abnormal metabolic effects of vitamin A were transferable to the testicular cells through the circulating blood, which contributed to abnormal spermatogenesis, as confirmed by FMT.These findings define a starting point for linking the testicular function and regulation of gut microbiota via host metabolomes and will be of potential value for the treatment of male infertility in MetS.