Objective To explore the protective effect of miR⁃320 on intestinal mucosal injury after burn in rats，and whether its mechanism is related to the inhibition of TLR4/NF⁃κB signaling pathway. Methods SD rats were randomly divided into 4 groups（n = 12）：sham group，sham+miR⁃320 group，scald group and scald+ miR⁃320 group.In the scald and scald+miR⁃320 groups，the Walker⁃Mason burn model was established. One week before the model was established，AAV⁃miR⁃320 was injected into the tail veins of the scald+miR⁃320 and sham+ miR⁃320 groups. To assess intestinal permeability，FITC⁃dextran assays were used.RT⁃qPCR was used to evaluate the expression of miR ⁃320 gene，and the protein levels of Occludin，ZO ⁃1 and TLR4/NF ⁃ κB signaling pathway were evaluated by Western blot. NCM460 cells were transfected with an miR ⁃ 320 mimics before LPS treatment， and the TNF⁃α and IL⁃6 cytokines levels were measured. Results The intestinal mucosa villi in the scald group were edematous，shorter and wider than in the sham group，the intestinal mucosa was significantly damaged at 24 hours，and there was a large number of inflammatory cell infiltration.In addition，burns increased DAO and FITC⁃ dextran levels and reduced miR⁃320，Occludin and ZO⁃1 expression in the intestinal tissues. Rats injected with the miR⁃320 mimic recovered from the damages.，and the expression of TLR4，MyD88 protein and p⁃NF⁃κB（p⁃p65） phosphorylation were significantly reduced（P < 0.05）. In vitro experiments confirmed that TLR4 is a potential target gene of miR⁃320，and miR⁃320 upregulation reversed the activation of the TLR4/NF⁃κB pathway induced by LPS in NCM460 cells，as well as the concentrations of TNF⁃αand IL⁃6 in the culture medium. Conclusion The level of miR⁃320 is reduced in burn⁃induced intestinal barrier dysfunction. By suppressing the TLR4/MyD88/NF⁃B signaling pathway，miR⁃320 upregulation reduces inflammation and improves intestinal barrier function.