When magnetic nanoparticles (MNPs) are used as a contrast agent for magnetic particle imaging (MPI), they are rapidly excreted during systemic circulation by the reticuloendothelial system such as Kupffer cells in the liver. Therefore, when considering clinical applications of MPI such as long-term monitoring of cardiovascular diseases, increasing the blood circulation time of MNPs by encapsulating the MNPs into actual cells such as red blood cells (RBCs) as a carrier may be practical. The purpose of this study was to create a biocompatible tracer for MPI by encapsulating MNPs (Resovist^®) into RBCs, and to investigate the effect of the encapsulating procedure on the properties of the RBCs loaded with MNPs (L-RBCs). MNPs were encapsulated into RBCs by the hypotonic dialysis method using a hypotonic buffer solution at three different osmotic pressures. Transmission (TEM) and scanning electron microscopic (SEM) images of the L-RBCs were obtained, and the saturation magnetic moment (Ms) was measured using vibrating sample magnetometry (VSM). From the response to a permanent magnet, the findings on TEM images, and the Ms values measured by VSM, we confirmed that RBCs were successfully loaded with MNPs using the hypotonic dialysis method. When the osmotic pressure was 80 mOsm, MNPs were not retained sufficiently inside the RBCs but were adhered to the membrane surface (TEM images), and RBCs lost their biconcave disc shape and shrunk after resealing (SEM images). At 160 mOsm, the Ms value was much lower than those at 80 and 120 mOsm. At 120 mOsm, the shape of RBCs was preserved (SEM images) and the Ms value was the highest when 0.2 ml (5.6mg of Fe) of Resovist^® was added to the dialysis tube for loading. In conclusion, we successfully encapsulated MNPs into RBCs using the hypotonic dialysis method. Our results suggest that an osmotic pressure of 120 mOsm is optimal for encapsulating MNPs into RBCs using the hypotonic dialysis method.