Orange juice-derived nanovesicles (ONVs) have significant biological activity and may be utilized to prevent high-fat diet-associated gut dysbiosis. In this study, ONVs were isolated and characterized. They were then orally administered at three different doses in a high-fat diet-induced bile acids metabolic disorder mouse model to determine their effects on gut microbiota-bile acids metabolism. Our results showed that oral intake of ONVs reshaped the gut microbiota and its associated bile acids metabolism in high-fat diet mice by increasing microbial diversity, beneficial bacteria abundance, secondary bile acid concentration, and decreasing taurine-conjugated bile acids content. These findings support a new diet-based strategy utilizing ONVs to prevent metabolic diseases targeting bile acid metabolism.