To determine whether and how probiotic supplement can alter gut microbiota dysbiosis and lipid metabolism disorders caused by perfluorobutanesulfonate (PFBS), the present study exposed adult zebrafish to 0, 10, and 100 μg/L PFBS for 28 days, with or without dietary administration of probiotic Lactobacillus rhamnosus. Regarding intestinal health and gut microbiota, probiotic supplement altered the innate toxicities of PFBS, depending on exposure concentration and the sex of the fish. Lactobacillus genus correlated positively (P < 0.001; r > 0.5) with other beneficial bacteria in the gut microbiota, thereby indirectly regulating host metabolic activities. In female fish, the PFBS and probiotic combination enhanced fatty acid synthesis and β-oxidation, but mitigated the accumulation of cholesterol in the blood compared with PFBS single exposure, highlighting the benefits of the probiotic to host health. In male zebrafish, probiotic administration antagonized the PFBS-induced disturbances of bile acid metabolism, presumably via farnesoid X receptor signaling. However, coexposure to PFBS and probiotic caused significant accumulation of triglyceride in male livers (2.6-fold relative to the control), implying the induction of hepatic steatosis. Overall, the present study underlined the potential of probiotics to modulate gut microbial dysbiosis and lipid metabolism disorders caused by PFBS exposure, which could provide implications to the application of probiotics in aquaculture. Copyright © 2020 American Chemical Society.