Xanthones are a family of compounds with several known biological activities and therapeutic potential for which information on their interaction with membrane transporters is lacking. Knowing that P-glycoprotein (P-gp) acts as a cellular defense mechanism by effluxing its toxic substrates, the aim of this study was to investigate the potential of five dihydroxylated xanthones as inducers of P-gp expression and/or activity and to evaluate whether they could protect Caco-2 cells against the cytotoxicity induced by the toxic P-gp substrate paraquat (PQ). After 24 h of incubation, all tested xanthones caused a significant increase in both P-gp expression and activity, as evaluated by flow cytometry using the UIC2 antibody and rhodamine 123, respectively. Additionally, after a short 45-min incubation, all the tested xanthones induced a rapid increase in P-gp activity, indicating direct pump activation without increased P-gp protein expression. The tested compounds also increased P-gp ATPase activity in MDR1-Sf9 membrane vesicles, demonstrating to be P-gp substrates. Moreover, when simultaneously incubated with PQ, all xanthones significantly reduced the cytotoxicity of the herbicide, and these protective effects were completely reversed upon incubation with a specific P-gp inhibitor. In silico studies evaluating the interactions between xanthones and P-gp in the presence of PQ suggested that a co-transport mechanism may be operating. A quantitative structure-activity relationship model was developed and validated, and the maximal partial charge for an oxygen atom was the descriptor predicted as being implicated in P-gp activation by the dihydroxylated xanthones. These results disclose new perspectives in preventing PQ- and other P-gp substrates-induced poisonings.