Co-contamination of arsenic (As) and fluoride (F) is widely distributed in groundwater, which are known risk factors for the nephrotoxicity. Emerging evidence has linked environmentally associated nephrotoxicity with the disturbance of gut microbiota and blood metabolites. In this study, we generated gut microbiota and blood metabolomic profile and identified multiple serum metabolites and gut bacteria species, which were associated with kidney injury on rat model exposed to As and F alone or combined. Combined As and F exposure significantly increased creatinine level. Abnormal autophagosomes and lysosome were observed, and the autophagic genes were enhanced in kidney tissue after single and combined As and F exposure. The metabolome data showed that single and combined As and F exposure remarkably altered the serum metabolites associated with the proximal tubule reabsorption function pathway, with glutamine and alpha-ketoglutarate level decreased in all exposed group. Furthermore, phosphatidylethanolamine (PE), the key contributor of autophagosomes, was decreased significantly in As and F + As exposed groups during the screen of autophagy-animal pathway. Multiple altered gut bacterial microbiota at phylum and species levels post As and F exposure were associated with targeted kidney injury, including p_Bacteroidetes, s_Chromohalobacter_unclassified, s_Halomonas_unclassified, s_Ignatzschineria_unclassified, s_Bacillus_subtilis, and s_Brevundimonas_sp._NA6. Meanwhile, our analysis indicated that As and F co-exposure possessed an interactive influence on gut microbiota. In conclusion, single or combined As and F exposure leads to the disruption of serum metabolic and gut microbiota profiles. Multiple metabolites and bacterial species are identified and associated with nephrotoxicity, which have potential to be developed as biomarkers of As and/or F-induced kidney damage.