〔Abstract〕 Objective To investigate the effect of mtDNA on monolayer permeability of Caco-2 cells and its mechanism. Methods The Caco-2 cell model was prepared by using Transwell culture plate. The mtDNA was extracted from the liver of C57 BL/6 J mice. Then, different concentrations of mtDNA(0, 1, 5, 10 μg/ml) and FITC-dex-tran were added to the top of the Transwell culture plate to detect the changes of TEER and FITC-dextran content in the bottom chamber of the Transwell culture plate at different time,which determined the best mtD NA concentration. Then,the experiment was randomized into four groups: FITC-dextran,mtD NA + FITC-dextran,TLR9 antagonists( ODN TTAGGG) + FITC-dextra,and mtD NA + ODN TTAGGG + FITC-dextran,and the changes of TEER and FITC-dextran content in the bottom chamber of the Transwell culture plate were detected at different time.Meanwhile,Caco-2 cells were pretreated with mtD NA,ODN TTAGGG,mtD NA + ODN TTAGGG,respectively.The expression of TLR9 mR NA was detected by q RT-PCR,and the levels of TNF-lα,IL-6,IL-1β and IL-18 in the supernatant were detected by ELISA. Results Compared with the control group,with the prolongation of the action time and the increase of the concentration of mtD NA,the TEER of the monolayer of Caco-2 cells decreased and the content of FITC-dextran in the bottom chamber increased,and the TEER decreased significantly when the concentration of mtD NA was 10 μg/ml. Compared with 10 μg/ml mtD NA group,the addition of ODN TTAGGG significantly reduced the falling speed of TEER,and reduced the content of FITC-dextran in the bottom chamber,meanwile,the expression of TLR9 mR NA and the levels of inflammatory factors also declined. Conclusion The mtD NA significantly increases the permeability of Caco-2 monolayer,which may be related to the activation of TLR9 signal pathway.