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Objective To investigate the effects of berberine on intestinal glucose absorption and the underlying mechanisms. Methods Eight-ten weeks old male Sprague-Dawley rats were divided into control group and berberine group. Blood glucose, serum insulin and glucagon-like peptide-1 (GLP-1) were tested in oral glucose tolerance test (OGTT) and intraperitoneal glucose tolerance test (IPGTT) after berberine gavage. Glucose transport test in vitro was applied to determine the specific target glucose transporter of berberine. Jejunum segments were treated with glucose (100 mmol/L), berberine (25, 50, 100, 200 μmol/L), glucose transporter 2 (GLUT2) inhibitor phloretin and sodium/glucose co-transporter 1 (SGLT-1) inhibitor phloridzin, 2-(N-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl) amino)-2-deoxyglucose (2-NBDG) was used to test the intestinal glucose transport ability. The expression and localization of GLUT2 and SGLT1 on intestinal epithelial cells membrane was tested by using Western blotting. Dunnett-t test was used to evaluate the statistical significance of difference between groups. Results Compared with control group, blood glucose was significantly decreased at 15, 30, and 60 min in berberine group in OGTT (t=2.812, 2.648, 3.044, all P<0.05), however, there was no significant difference between two groups in IPGTT results. Compared with control group, serum insulin was significantly decreased at 15, 30, 60, and 120 min in berberine group (t=3.981, 3.583, 2.658, 2.707, all P<0.05), and serum GLP-1 level showed no significant difference between the two groups. Compared with glucose group (1.00±0.03), berberine (25, 50, 100, 200 μmol/L) treatment reduced glucose transport significantly (0.84±0.05, 0.62±0.04, 0.47±0.04, 0.46±0.04 respectively, q=2.847, 7.067, 9.712, 9.786, all P<0.05). Furthermore, there was no significant difference in glucose transport between the phloretin group and berberine+phloretin group (0.60±0.02 vs 0.58±0.02, q= 0.68, P>0.05), but glucose transport decreased significantly in berberine+phloridzin group when compared with that in phloridzin group (0.42±0.02 vs 0.68±0.03, q=7.436,P<0.05). Western blotting showed that intestinal epithelial cells membrane GLUT2 localization decreased significantly in berberine group than that in control group (0.57±0.04 vs 1.00±0.00, t=9.808, P<0.05). Conclusion Berberine decreases intestinal glucose absorption through inhibiting intestinal epithelial cells membrane GLUT2 translocation. [ABSTRACT FROM AUTHOR]