Aristolochic acid (AA) can accumulate in the tubulointerstitium and cause kidney-specific injuries. However, the mechanism by which AA induces nephropathy remains largely unknown. This study explored the effect of AA-I on tight junctions (TJs), and the fence function in a renal epithelial cell (REC). NRK-52E cells were exposed to different concentrations of AA-I for 4 h or 25 μM AA-I for different time. Cell viability was detected by MTT, cell apoptosis by flow cytometric analysis, the expression of zonula occludens-1 (ZO-1), E-cadherin and polarity scaffold (Par3) by western blot and immunofluorescence, cell membrane permeability by transepithelial electrical resistance (TEER). It was found that AA-I reduced the expression of ZO-1, E-cadherin, and Par3 in a concentration- and time-dependent fashion, and altered the distribution of ZO-1 and Par3 from cell membrane to cell plasma. In parallel to the reduced expression of TJ proteins, TEER exhibited a significant reduction in response to AA-I treatment in a time- and concentration-dependent manner. Meanwhile, α-SMA expression in cells was increased following AA-I treatment. In contrast, cell viability and apoptosis were unaltered with the doses of AA-I tested. Our findings show for the first time that AA-I treatment in cultured RECs induced a rapid disruption of TJ and the fence function preceding apoptosis, which indicated that aberrant expression of TJ proteins within RECs may be involved in initiating the renal tubulointerstitial disorders.