Abstract: Background and Objective: Intestinal barrier dysfunction is associated with many clinical diseases. However, no efficient biomarkers and therapeutic drugs of intestinal barrier dysfunction have been found till now. In this study, the positive effects of matrine on intestinal barrier via SPRY4-IT1 was investigated. Materials and Methods: Expression of tight junction associated proteins was determined through SPRY4-IT1 inhibition or overexpression Caco-2 cells. DSS-induced mice colitis was used to verify the protective role of matrine on intestinal barrier and the regulation on SPRY4-IT1. SPRY4-IT1 and related was detected in clinical specimens of acute stercoral obstruction patients. Results: QRT-PCR showed that SPRY4-IT1 inhibitor could prohibit the role of matrine on intestinal barrier protection. The intestinal permeability of DSS-induced mice colitis was increased and the tight junction-associated proteins were decreased, which could be relieved by matrine. There was also a lower of SPRY4-IT1 in clinical colonic samples in stercoral obstruction patients. Conclusion: Matrine could protect the intestinal barrier from dysfunction via the signaling path way of SPRY4-IT1.