We report anatomically correct 3D-printed mouse phantoms that can be used to plan experiments and evaluate analysis protocols for magnetic particle imaging (MPI) studies. The 3D-printed phantoms were based on the Digimouse 3D whole body mouse atlas and incorporate cavities representative of a liver, brain tumor, and orthotopic breast cancer tumor placed in anatomically correct locations, allowing evaluation of the effect of precise doses of MPI tracer. To illustrate their use, a constant tracer iron mass was present in the liver for the breast (200 μg) and brain tumor (10 μg) model, respectively, while a series of decreasing tracer iron mass was placed in the tumor region. MPI scans were acquired in 2D and 3D high sensitivity and high sensitivity/high resolution (HSHR) modes using a MOMENTUM imager. A thresholding algorithm was used to define regions of interest (ROIs) in the scans and the tracer mass in the liver and tumors was calculated by comparison of the signal in their respective ROI against that of known mass fiducials that were included in each scan. The results demonstrate that this approach to image analysis provides accurate estimates of tracer mass. Additionally, the results show how the limit of detection in MPI is sensitive to the details of tracer distribution in the subject, as we found that a greater tracer mass in the liver cavity resulted in poorer sensitivity in tumor regions. These experiments illustrate the utility of the reported 3D-printed anatomically correct mouse phantoms in evaluating methods to analyze MPI scans and plan in vivo experiments.