Abstract Background Disruption of intestinal barrier function and an imbalance in intestinal immunity are crucial for the occurrence and development of ulcerative colitis. Because of their important roles in regulating inflammation and immunity, exosomes (Exos) released from bone marrow mesenchymal stem cells (BMSCs) may be useful for treating ulcerative colitis. The EphB/EphrinB signaling pathway plays a crucial role in the inflammatory process and the development and function of immune cells, and can mediate long-distance intercellular communication through extracellular vesicles. This study was conducted to explore the effects of pre-modified BMSC-Exos expressing EphB2 (EphB2-Exos) on immunoregulation in vitro. Methods We transfected a lentivirus vector encoding EphB2 into BMSCs and isolated EphB2-Exos from the culture supernatant. Inflammation and oxidative damage in the human colon adenocarcinoma cell line (Caco-2) were induced by dextran sulfate sodium/hydrogen peroxide. In addition, spleen CD4+ T lymphocytes of rats were sorted in vitro. We conducted a series of experiments to explore the biological functions of EphB2-Exos. Results EphB2-Exos were successfully isolated and were found to significantly protect the activity, proliferation, and migration of Caco-2 cells that were inhibited by dextran sulfate sodium. EphB2-Exos alleviated inflammation and apoptosis and increased the activity of antioxidant enzymes while inhibiting oxidative stress in Caco-2 cells. EphB2-Exos restored barrier function by inhibiting the RhoA/ROCK pathway and regulated the polarization of CD4+T cells. Conclusion EphB2-Exos enhanced intestinal barrier function and regulated the immune balance by inhibiting the RhoA/ROCK pathway in vitro. These findings suggest that EphB2-Exos can be applied as a cell-free therapy for ulcerative colitis.