The present study aimed to explore the role of CXCR4 and protein C system (PCS) in the experimental ulcerative colitis (UC). The expression of CXCR3, CCR10, and CXCR4 in dextran sulfate sodium (DSS)-induced colitis mouse model was measured by immunohistochemistry and western blot analysis. In vitro studies with microvascular endothelial cells (MVECs) were performed. The expression of endothelial protein C receptor (EPCR) and thrombomodulin (TM) were detected by RT-PCR and western blot analysis. Activities of protein C (PC), protein S (PS), activated PC (APC) were evaluated in cells pre-treated with JNK inhibitor SP600125 and c-Jun silencing. DSS mice showed up-regulated expression of CXCR4, higher macroscopic score and histological score (P<0.05), as well as elevated levels of SDF-1α (P<0.05) compared with wild type, CXCR4-/-, or CXCR4-/- +DSS mice. In DSS mice, EPCR expression was down-regulated (P<0.05), accompanied by decreased activity of PC and PS (P<0.05 or P<0.01) with an up-regulated expression of pJNK MAPK and pc-Jun (P<0.05). Moreover, the macroscopic score and histological score index, SDF-1α levels, EPCR expression, PC activity, pJNK, and pc-Jun were reversed in CXCR4-/- +DSS mice (P<0.05). In vitro, SDF-1α-induced inhibition of the PCS was blunted by SP600125 (P<0.05). Meanwhile, down-regulation of c-Jun rescued the inhibition of PCS (P<0.05). MVECs with retrovirus-mediated transfection of c-Jun demonstrated a strong trans-inactivation effect on the EPCR promoter (P<0.05). These findings suggest that CXCR4 is involved in UC pathogenesis and could be a promising therapeutic target for UC treatment.