BACKGROUND AIMS: The purpose of the study was to investigate the feasibility of in vitro and in vivo bioluminescence imaging (BLI), fluorescence imaging (FI) and magnetic resonance imaging (MRI) to trace transplanted bone mesenchymal stromal cells (BMSCs) labeled with the firefly luciferase (Fluc) reporter gene, CyI dyes and ultra-small super-paramagnetic iron oxide (USPIO) particles. METHODS: Fluc-transfected BMSCs were further labeled with CyI dyes and USPIO particles, respectively. Acute myocardial infarction models of different weighted Sprague-Dawley rats and Balb/c mice were established, and BLI and FI were performed in vivo and ex vivo to determine the optimal method of optical imaging. Finally, BLI and MRI were selected to trace transplanted BMSCs in a murine model in vivo. RESULTS: BLI was found to be the optimal optical imaging method in vivo, compared with FI, and mice were found to be the optimal animal model, compared with rats. A significant BLI signal intensity was detected in the heart region in the BMSC-treated mice group (40,552 ± 6073 counts, n = 26) and gradually decreased below the detection threshold. Two distinct hypo-intense regions were observed in the anterior wall of the heart, where stem cells were injected on MR images obtained with the gradient recalled echo cine sequence in the BMSC-treated mice group. CONCLUSIONS: Transplanted BMSCs labeled with Fluc reporter gene and USPIO particles can be traced with the use of BLI and MRI in a mouse model of acute myocardial infarction.