Understanding the effects of magnetic iron nanoparticles (MINPs) on the immune response is vitally important for biomedical applications such as cancer therapy, disease diagnosis and novel cancer imaging. In this study, lipid modified MINPs were designed and prepared by introducing the neutral lipid DSPE-PEG or the zwitterionic lipid DSPE-PCB into hydrophobic MINPs through hydrophobic interaction (L-MINPs and ZL-MINPs, respectively). The effect of L-MINPs and ZL-MINPs on the intracellular accumulation and immune responses of three kinds of antigen processing cells was examined. The results indicated that the high cellular uptake efficiency of surface coated MINPs was strongly related to the nature of the coating lipid, with the zwitterionic lipid being more effective than PEGylated ones. Besides, the results from flow cytometry (FCM), confocal laser scanning microscopy (CLSM) and Prussian blue staining demonstrated a time- and concentration-dependent MINP internalization. The uptake of zwitterionic lipid modified MINPs (ZL-MINPs) induced very low cytotoxicity and a strong mixed Th1/Th2 type immune response. L-MINPs could induce a strong increase in pro-inflammatory cytokines with a slight secretion of Th2 cytokines. Besides, no IL-10 was observed in both groups, indicating that MINPs with lipid modification were absence of immunosuppression. In conclusion, this study addresses an important implication of the lipid type and Fe concentration on the immune stimulation of cells and supports the potential for further development of biomedical applications.