AIM: To characterize the intestinal transport and mechanism of metformin in rats and to investigate whether or not metformin is a substrate for P-glycoprotein (P-gp).METHODS: The effective intestinal permeability of metformin was investigated using single-pass intestinal perfusion (SPIP) technique in male Waster rats. SPIP was performed in three isolated intestinal segments (duodenum, jejunum and ileum) at the same concentration of metformin (50 μg/mL) to test if the intestinal transport of metformin exhibited sitedependent changes, and in a same isolated intestinal segment (duodenal segment) at three different concentrations of metformin (10, 50, 200 μg/mL) to test if the intestinal transport of metformin exhibited concentration-dependent changes. Besides, P-gp inhibitor verapamil (400 μg/mL) was co-perfused with metformin (50 μg/mL) in the duodenum segment to find out if the intestinal absorption of metformin was affected by P-gp exiting along the gastrointestinal track.Stability studies were conducted to ensure that the loss of metformin could be attributed to intestinal absorption.RESULTS: The effective permeability values (Peff) of metformin in the jejunum and ileum at 50 μg/mL were significantly lower than those in the duodenum at the same concentration. Besides, Peff values in the duodenum at high concentration (200 μg/mL) were found to be significantly lower than those at low and medium concentrations (10 and 50 μg/mL). Moreover the coperfusion with verapamil did not increase the Peff value of metformin at 50 μg/mL in the duodenum.CONCLUSION: Metformin could be absorbed from the whole intestine, with the main absorption site at duodenum. This concentration-dependent permeability behavior in the duodenum indicates that metformin is transported by both passive and active carrier-mediated saturable mechanism. The Peff value can not be increased by co-perfusion with verapamil, indicating that absorption of metformin is not efficiently transported by P-gp in the gut wall. Furthermore metformin is neither a substrate nor an inducer of P-gp. Based on the Peff values obtained in the present study and using established relationships,the human fraction dose absorbed for metformin is estimated to be 74%-90% along human intestine.