Endothelial progenitor cells (EPCs) have been used in clinical trials for the treatment of patients with ischemic diseases, such as myocardial infarction and limb ischemia, as they could secrete angiogenic growth factors and promote neovascularization. However, the clinical employment of EPC therapy relies on the establishment of a noninvasive, timely, effective, and safe assessment system. In this study, we recruited superparamagnetic iron oxide nanoparticles (SPION) enhanced magnetic resonance imaging (MRI) to track EPCs in vitro. We evaluated the labeling efficiency of SPION in EPCs without cationic transfection reagents and determined the threshold value of the labeled cell quantity for MRI. Our data suggested that 1×105 EPCs incubated with SPION at a concentration of 100 μg Fe/mL for 24 h could be detected with a clinical 3T MRI T₂ scan. SPION were not cytotoxic for EPCs; however, SPION induced autophagy and impaired neovascularization. We then examined if the impairment could be alleviated through the inhibition of autophagy. The observed autophagy response and its function in EPCs may provide a strategy to improve the safety of SPION labeling. It may also verify that the assessment of autophagy may be a more sensitive detection method to evaluate the biocompatibility of tracking reagents.