The purpose of this study was to evaluate the effectiveness of intracellular magnetic hyperthermia treatment (MHT) in comparison with that of extracellular MHT using magnetic particle imaging (MPI). Colon-26 cells were implanted subcutaneously into the backs of 8-week-old male BALB/c mice. When the tumor volume reached approximately 100 mm3, the mice were divided into control (n = 10), extracellular MHT (n = 8), and intracellular MHT groups (n = 7). In the control group, MHT was not performed. In the extracellular MHT and intracellular MHT groups, the tumors were injected directly with magnetic nanoparticles (MNPs) (400 mM Resovist®) and were heated for 20 min using an alternating magnetic field. During MHT, the temperatures of the tumor and rectum were measured using optical fiber thermometers. In the extracellular MHT group, MHT was performed 15 min after the injection of MNPs, whereas MHT was performed one day after the injection of MNPs in the intracellular MHT group. In both groups, MPI images were obtained using our MPI scanner immediately before, immediately after, and 7 and 14 days after MHT. After the MPI studies, we drew a region of interest (ROI) on the tumor in the MPI image and calculated the average, maximum, and total MPI values and the number of pixels within the ROI. Transmission electron microscopic (TEM) images were also obtained from resected tumors. In all groups, tumor volume was measured every day and the relative tumor volume growth (RTVG) was calculated. The TEM images showed that almost all the MNPs were aggregated in the extracellular space in the extracellular MHT group, whereas they were contained within the intracellular space in the intracellular MHT group. Although the temperature of the tumor in the intracellular MHT group was significantly lower than that in the extracellular MHT group, the RTVG value in the intracellular MHT group was significantly lower than that in the control group 2 days or more after MHT and that in the extracellular MHT group 3, 4, and 5 days after MHT. The average MPI value normalized by that immediately before MHT in the intracellular MHT group was significantly higher than that in the extracellular MHT group immediately and 7 days after MHT. The maximum and total MPI values normalized by those immediately before MHT in the intracellular MHT group were significantly higher than those in the extracellular MHT group 7 days after MHT, suggesting that the temporal change of MNPs within the tumor in the intracellular MHT group was smaller than that in the extracellular MHT group. Our results suggest that intracellular MHT is more cytotoxic than extracellular MHT in spite of a lower temperature rise of tumors, and that MPI is useful for evaluating the difference in the temporal change of MNPs in the tumor between extracellular MHT and intracellular MHT.