Objectives: Bioactivation of food contaminants and drugs by CYP3A4 in liver and intestine is major concern for their safety evaluation in human. Hepatic bioactivation is assessed by in vitro methods such as liver microsomes and hepatocytes, whereas in vitro method to evaluate intestinal bioactivation have never been developed. Although Caco-2 cells are widely used as an in vitro human intestinal model, their CYP3A4 activity is much lower than that in the human intestine. A doxycycline-inducible CYP3A4-expressing Caco-2 cell line was established in the Mizuguchi Lab (Sci Rep, 11, 11670, 2021). This cell line exhibits CYP3A4 activity comparable to that found in the adult intestine. In this study, we utilized the CYP3A4-expressing cells to evaluate the intestinal CYP3A4-dependent bioactivation of xenobiotics.Methods: Acetaminophen (APAP), and aflatoxin B1 (AFB1) were used as xenobiotics metabolically activated by CYP3A4. Cytotoxicity, barrier integrity and epithelial permeability were investigated in the inducible CYP3A4-expressing Caco-2 cell line.Results: Upon APAP treatment, there is no significant difference between induced and uninduced Caco-2 cells in cytotoxicity and barrier integrity. However, cellular toxicity of AFB1 in induced Caco-2 cells was approximately 5-fold that of uninduced cells. Exposure to AFB1 diminished barrier integrity while uninduced cells remained stable. Hence, there was a notable increase in paracellular permeation of 4- and 20-kDa dextran in induced Caco-2 cells, reaching levels 5.4- and 5.2-fold higher than those in uninduced cells.Discussion: CYP3A4-dependent bioactivation may be a risk for transient disruption of intestinal barrier, followed by the permeation of other macromolecules from intestinal fluids into the body. When evaluating food safety, it is important to consider the evaluation of intestinal barrier function. The CYP3A4-expressing Caco-2 cells can be a valuable cellular model for intestinal safety evaluation.