Downregulation of P-glycoprotein (P-gp) is implicated in the pathophysiology of inflammatory bowel disease (IBD). Berberine, a principal isoquinoline alkaloid extracted from Berberis species, has been reported to exhibit therapeutic potential in IBD. In this study, we used a dextran sulfate sodium (DSS)-induced colitis rat model to evaluate the effect of berberine on P-gp and explore its mechanism of action. Berberine treatment improved DSS-induced colitis symptoms, attenuated inflammatory markers (myeloperoxidase, tumor necrosis factor-a, and interleukin-1b and -6), and enhanced P-gp expression in a dose-dependent manner. Although colonic expression of the P-gp–related nuclear receptor pregnane X receptor and transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) were downregulated in the colitis model, gene and protein expression analysis revealed that berberine treatment reversed only the downregulation of Nrf2. In vitro studies using Caco-2 cells showed that the multidrug resistance 1 (MDR1) gene and P-gp protein were upregulated by berberine in a dose- and time-dependent manner. Significant upregulation of the MDR1 gene by berberine was abrogated by Nrf2 silencing, indicating that the Nrf2-mediated pathway was responsible for this activation. Luciferase assays showed a dose-dependent increase in Nrf2 reporter gene activity after berberine treatment in Caco-2 cells, with a significant 2-fold elevation at 2.5 mM berberine, suggesting that berberine is a strong Nrf2 activator. These results indicate the possible involvement of Nrf2-mediated upregulation of P-gp in the therapeutic effect of berberine on colitis and highlight the potential of P-gp and/or Nrf2 as new therapeutic targets for IBD.