Increased tight junction (TJ) barrier permeability, induced by tumour necrosis factor (TNF)‐α, may lead to the defects in TJ barrier and subsequent development of inflammation. Recent evidence suggests that miR‐21 is implicated in inflammatory diseases. However, the physiological role of miR‐21 in intestinal permeability remains elusive. This study aimed to explore the role of miR‐21 in intestinal epithelial tight junction permeability. The filter‐grown Caco‐2 monolayers model system was established to mimic intestinal barrier defect. The tight junction proteins were detected by immunofluorescence and western blot analysis. The expression of miR‐21 was assessed by real‐time polymerase chain reaction (PCR). We found that the expression of miR‐21 was increased significantly in TNF‐α induced intestinal TJ barrier defect model. miR‐21 overexpression significantly enhanced while miR‐21 knockdown significantly decreased intestinal permeability. In addition, miR‐21 overexpression significantly increased while miR‐21 knockdown significantly decreased the levels of interleukin‐6, interleukin‐8 and prostaglandin E2 in cell culture medium. Furthermore, miR‐21 positively regulated Akt phosphorylation and negatively regulated Phosphatase and tensin homolog (PTEN) expression in Caco‐2 cells. Our results suggest that miR‐21 may regulate intestinal epithelial tight junction permeability through PTEN/PI3K/Akt signalling pathway. This promotes the feasibility of targeting miR‐21 in the clinical to preserve the intestinal barrier. Copyright © 2015 John Wiley & Sons, Ltd.