Purpose The presence of tumor-associated macrophages (TAMs) in breast cancer correlates strongly with poor outcome. The purpose of this study was to develop a clinically applicable, non-invasive diagnostic assay for selective targeting and visualization of TAMs in breast cancer, based on magnetic resonance (MR) imaging and clinically applicable iron oxide nanoparticles. Experimental Design F4/80-negative mammary carcinoma cells and F4/80-positive TAMs were incubated with iron oxide nanoparticles and were compared regarding MR signal changes and iron uptake. MMTV-PyMT transgenic mice harboring mammary carcinomas underwent nanoparticle-enhanced MR up to 1 hour (h) and at 24 h post injection (p.i.). The tumor enhancement on MR images was correlated with the presence and location of TAMs and nanoparticles on confocal microscopy. Results In vitro studies revealed that iron oxide nanoparticles are preferentially phagocytosed by TAMs, but not by malignant tumor cells. In vivo, all tumors demonstrated an initial contrast agent perfusion on immediate postcontrast MR images with gradual transendothelial leakage into the tumor interstitium. At 24 h p.i., all tumors demonstrated a persistent signal decline on MR scans. TAM-depletion via αCSF1 mAb lead to significant inhibition of tumor nanoparticle enhancement. Detection of iron using DAB-enhanced Prussian Blue staining, and immunodetection of CD68 localized iron oxide nanoparticles to TAMs, indicating that the MR signal effects on delayed MR images were largely due to TAM-mediated uptake of contrast agent. Conclusion These data indicate that tumor-enhancement with clinically applicable iron oxide nanoparticles may serve as a new biomarker for long-term prognosis, related treatment decisions and the evaluation of new immune-targeted therapies.