P-glycoprotein (Pgp) is an ATP-dependent transmembrane protein involved in the efflux of lipophilic substances. The aim of this study was to evaluate the effect of oxidative stress on the transport of a Pgp substrate through the monolayer of Caco-2 cells overexpressing this transport protein. Oxidative stress was modeled by incubating the cells with H2O2. Exposure to H2O2 at concentrations of 10 and 50 μM for 3 h reduced the Pgp activity but not the content of Pgp, while the integrity of the cell monolayer did not change. The increase of the prooxidant concentration to 100 μM reduced the content of Pgp, violated the integrity of the cell monolayer, and increased the transcellular and paracellular transport of fexofenadine. A 24-h exposure to 0.1–1 µM H2O2 resulted in an increase in the content of Pgp mediated by the Nrf2 transcription factor, while the activity of the transport protein remained unchanged. At a prooxidant concentration of 10 µM, the Pgp activity decreased and the cell membrane permeability increased, while at concentrations of 50–100 µM, the content (100 µM) and activity of Pgp decreased, and the paracellular and transcellular permeability of the cell monolayer increased for fexofenadine, a substrate of the transport protein. Thus, H2O2 increased the transport of the Pgp substrate fexofenadine through the cell monolayer by inhibiting the activity of the transport protein, reducing its content, as well as violating the integrity of the cell membrane and intercellular contacts. The cells can adapt to these effects by increasing the content of Pgp.