Hepatic and extrahepatic tissues participate in xenobiotic detoxication, carcinogen activation, prodrug processing, and estrogen regulation through UDP-glucuronosyltransferases (UGTs/Ugts) and sulfotransferases (SULTs/Sults). Wild-type (WT) and efflux transporter knockout (KO) FVB mice have been commonly used to perform the studies of pharmacokinetics, metabolism, and toxicity. We employed the developed UHPLC-MS/MS approach to gain systematic insight on gender-specific of Ugts and Sults in major metabolic organs. Results showed that the liver was the most abundant with Ugts/Sults, followed by the small intestine and the kidney. In the liver, Ugt2b5, Ugt2b1, Ugt1a6a, Ugt1a1, Sult1a1, and Sult1d1 were the major isoforms. The protein amounts of Ugt1a9 were significantly higher in male efflux transporter KO mice than in WT mice, whereas Ugt1a5 and Sult1a1 severely decreased in female efflux transporter KO mice. In WT and efflux transporter KO mice, the expression levels of Ugt1a1, Ugt1a5, Sult1a1, Sult1d1, and Sult3a1 were female-specific, whereas those of Ugt2b1, Ugt2b5, and Ugt2b36 were male-specific. In the small intestine, Ugt1a1, Sult1b1, and Sult2b1 were the major isoforms. The protein levels and gender differences of Ugts/Sults were obviously affected when KO of Mdr1a, and Bcrp1, Mrp1, Mrp2, and Mdr1a, respectively. The KO of efflux transporter affected the protein amounts of Ugts/Sults in the kidney, heart, and spleen. Therefore, a better understanding of the expression profiles and gender-specific of Ugts and Sults in major metabolic organs of WT and efflux transporter KO mice is useful for the evaluation of potential efficacy, and toxicity of corresponding substrates.