Sepsis is a severe organ dysfunction syndrome caused by the host's dysfunctional response to infection. Sepsis can severely damage intestinal epithelial tissue, lead to intestinal barrier dysfunction, and seriously endanger human health. Therefore, this study aimed to explore the mechanism of miR-20a in sepsis-induced intestinal barrier dysfunction. In this study, mice and NCM460 cells were subjected to cecal ligation and puncture (CLP) and 1 μg/mL LPS, respectively, to establish a sepsis model. The expression of relevant genes, apoptosis, inflammation, and intestinal barrier dysfunction-related indices under the conditions of overexpression of miR-20a or DUSP3 and knockdown of DUSP3 or OCLN were assessed by western blotting, RT-qPCR, ELISA, flow cytometry, immunofluorescence, and HE staining. The experimental results revealed that in sepsis-induced intestinal barrier dysfunction, the expression of miR-20a and OCLN was downregulated, whereas that of DUSP3 was upregulated. Functionally, miR-20a inhibited LPS-induced intestinal epithelial cell apoptosis and inflammation and relieved sepsis-induced intestinal barrier dysfunction in mice. Experiments investigating the downstream mechanisms revealed that miR-20a overexpression suppressed LPS-induced intestinal epithelial cell apoptosis and inflammation and relieved sepsis-induced intestinal barrier dysfunction by targeting and inhibiting DUSP3 levels and OCLN ubiquitination. In conclusion, miR-20a relieves sepsis-induced intestinal barrier dysfunction by inhibiting DUSP3 and suppressing the ubiquitination of OCLN.