BACKGROUND: The successful development of mesenchymal stromal cells (MSCs)-based therapeutic strategies in inflammatory bowel disease (IBD) hinges on the ability to assess the influence of the administration route, while also tracking the cells' in vivo location, distribution, and long-term viability. Moreover, understanding the biological fate of these cells-particularly their activation, differentiation, and interaction within the host environment-is crucial to optimising their therapeutic potential. METHODS: This study explores the use of magnetic particle imaging (MPI) with superparamagnetic iron oxide nanoparticles (SPIONs) to track MSCs in colitis mice. SPION-labelled MSCs were administered through intravenous (IV), intraperitoneal (IP), and intrarectal (IR) routes. MPI was employed to track MSCs. FINDINGS: MPI provided dynamic tracking of MSC distribution, viability, and homing, revealing that IP injections led to more targeted and sustained delivery to inflamed colon tissues, with higher cell survival and efficacy in modulating inflammation. Other administration routes also demonstrated specific distribution patterns, but with shorter retention times. The prolonged survival of MSCs following IP injection may be attributed to the immune environment in the peritoneal cavity, which significantly impacts MSCs, influencing their survival and migration. Further analyses showed that MSCs exposed to inflammatory peritoneal fluid exhibited enhanced homing capabilities, and IFN-gamma pretreatment significantly improved MSC retention in the inflamed colon, as detected by MPI/CT imaging. INTERPRETATION: Our findings, observed through MPI, highlight the potential of IP injection as a preferred method for MSC-based therapies, offering strategies to improve treatment outcomes specifically in IBD and potentially in other inflammatory conditions. FUNDING: The National Natural Science Foundation of China (62027901, 82372144), the Natural Science Foundation of Jiangsu Province (BK20230491), a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions.