Objective To investigate the effect of Linggui Zhugan decoction on intestinal mucosal barrier and immune balance in rats with ulcerative colitis（UC） by regulating the miR-140-5p/Toll like receptor 4（TLR4）/nuclear factor-kappa B（NF-κB） signaling pathway. Methods Sprague-Dawley rats were induced with 2，4，6-trinitrobenzene sulfonic acid to establish a model of UC，and then the rats were randomly divided into model group，low-dose Linggui Zhugan decoction group，high-dose Linggui Zhugan decoction group，NC-miR-140-5p（miR-140-5p negative control） group，and high-dose Linggui Zhugan decoction group+miR-140-5p antagonist（miR-140-5p inhibitor） group，with 10 rats in each group. Another 10 rats were selected as control group. After grouping and intervention，colonic mucosal injury index（CMDI） score and colon length were measured for each group； transmission electron microscopy was used to observe the ultrastructure of intestinal mucosal barrier；flow cytometry was used to measure the percentages of Treg and Th17 cells in peripheral blood and Treg/Th17 ratio；ELISA was used to measure the serum levels of immune inflammatory factors such as interleukin-6（IL-6），interleukin-17（IL-17），interleukin-10（IL-10），and transforming growth factor-β1（TGF-β1） in rats； quantitative real-time PCR and Western blotting were used to measure the expression of the factors associated with the miR-140-5p/TLR4/NF-κB signaling pathway in the colonic mucosal tissue of rats. Results Compared with the control group，the model group had severe damage of the ultrastructure of colonic mucosal barrier and significant increases in CMDI，the percentage of Th17 cells，the serum levels of IL-6 and IL-17，the mRNA and protein expression levels of TLR4 in colonic mucosal tissue，and p-NF-κB p65/NF-κB p65（P<0.05），as well as significant reductions in colon length，the percentage of Treg cells，Treg/Th17 ratio，the serum levels of IL-10 and TGF-β1，the protein expression levels of claudin-1 and claudin-4，and the expression of miR-140-5p in colonic mucosal tissue（P<0.05）. Compared with the model group，the low- and high-dose Linggui Zhugan decoction groups had alleviation of the damaged ultrastructure of colonic mucosal barrier and significant reductions in CMDI，the percentage of Th17 cells，the serum levels of IL-6 and IL-17，the mRNA and protein expression levels of TLR4 in colonic mucosal tissue，and p-NF-κB p65/NF-κB p65（P<0.05），as well as significant increases in colon length，the percentage of Treg cells，Treg/Th17 ratio，the serum levels of IL-10 and TGF-β1，the protein expression levels of claudin-1 and claudin-4，and the expression of miR-140-5p in colonic mucosal tissue（P<0.05）. The NC-miR-140-5p group had no significant changes in these indicators（P>0.05），and high-dose Linggui Zhugan decoction had a stronger effect. Downregulation of miR-140-5p could weaken the effect of high-dose Linggui Zhugan decoction on various indicators in a rat model of UC. Conclusion Linggui Zhugan decoction can inhibit TLR4/NF-κB signal activation by upregulating miR-140-5p，thereby promoting the shift of Treg/Th17 immune balance to Treg，inhibiting immune inflammation，and alleviating intestinal mucosal barrier damage in UC rats.