Manganese enhanced MRI (MEMRI) has been used to generate layer-specific contrast in preclinical neuroimaging studies, especially in the cerebellum. However, the cell types that contribute most to MEMRI signal have not yet been identified. In this study, we registered high resolution MEMRI to immunohistochemistry of the cerebellum to verify layer localization of signal. We found that the Purkinje cell layer (PCL) was the source of hyperintensity. Next, we manipulated cell types of the PCL using genetically engineered mouse models and quantified MEMRI signal changes. These results strongly suggest Purkinje cells are the primary cellular source of hyperintensity in cerebellar MEMRI.