Stem cell transplantation showed promising results in IBD management. However, the therapeutic impacts of cell delivery route that is critical for clinical translation are currently poorly understood. Here, three different MSCs delivery routes: intraperitoneal (IP), intravenous (IV), and anal injection (AI) were compared on DSS-induced colitic mice model. The overall therapeutic factors, MSCs migration and targeting as well as local immunomodulatory cytokines and FoxP3(+) cells infiltration were analyzed. Colitis showed varying degrees of alleviation after three ways of MSCs transplantation, and the IP injection showed the highest survival rate of 87.5% and displayed the less weight loss and quick weight gain. The fecal occult blood test on the day 3 also showed nearly complete absence of occult blood in IP group. The fluorescence imaging disclosed higher intensity of engrafted cells in inflamed colon and the corresponding mesentery lymph nodes (MLNs) in IP and AI groups than the IV group. Real time-PCR and ELISA also demonstrate lower TNF-α and higher IL-10, TSG-6 levels in IP group. The immunohistochemistry indicated higher repair proliferation (Ki-67) and more FoxP3(+) cells accumulation of IP group. IP showed better colitis recovery and might be the optimum MSCs delivery route for the treatment of DSS-induced colitis.