Background: Leonurine (Leo), a promising antilipemic agent that has been approved for clinical trials, is extensively metabolized into bioactive Leonurine-10-O-β-glucuronide (L-10-G) vivo.Objective: To explore the effects of breast cancer resistance protein (Bcrp) and multidrug resistance protein 2 (Mrp2) on the disposition of L-10-G. Methods: The pharmacokinetics, tissue distribution and intestinal perfusion of Leo were studied by using efflux transporter gene knockout mouse models. The enzyme kinetics via liver and intestinal microsomes were also examined. Results: After intravenous injection with Leo, the AUC0-∞ values of L-10-G in Bcrp1-/- and Mrp2-/- mice were 1.55-fold and 16.80-fold higher, respectively, than those in wild-type FVB mice (P < 0.05). After oral administration, the AUC0-∞ value of L-10-G showed a 2.82-fold increase in Mrp2-/- mice compared with wild-type FVB mice (P < 0.05). After gavage with Leo for 10 and 25 min, the bile accumulation of L-10-G in Mrp2-/- mice was 3-fold and 22-fold lower, respectively, than that in wild-type FVB mice (P < 0.05). Besides, the intestinal excreted amount of L-10-G showed 2.22-fold and 2.68-fold decrease in Bcrp1-/- and Mrp2-/- mice, respectively, compared with that in wild-type FVB mice (P < 0.05). The clearance of L-10-G decreased in liver microsomes and increased in intestinal microsomes of Bcrp1-/- and Mrp2-/- mice compared to the wild-type FVB mice (P < 0.05). Conclusion: Both Bcrp and Mrp2 are involved in the disposition of L-10-G, and Mrp2 exhibits a superior influence.