The magnetic properties of the antiferromagnetic cores in ferritin are of importance in the construction and improvement of ferritin-based magnetic resonance imaging systems and their application to environmental magnetism. In this study, we carry out integrated magnetic and transmission electron microscopy analyses of horse spleen ferritin (HoSF) to understand the relationships between the magnetic behavior of HoSF and temperature, applied field and grain-size distributions. The R-value from the Wohlfarth-Cisowski test for the investigated sample at 5 K was 0.46, indicating very weak magnetostatic interactions among the nanoparticles of HoSF. The nanoparticles of HoSF show superparamagnetic properties at room temperature, while below the blocking temperature of T b ≈ 12 K it has a net magnetic moment that comes from the uncompensated spins of the nanoparticle surface or spin-canting. The thermal relaxation process of HoSF follows the Néel-Arrhenius expression. From low-temperature AC susceptibility data, we calculated the effective magnetic anisotropy energy E a=(5.52±0.16)×10−21 J; the effective magnetic anisotropy energy constant K eff =(4.65±0.14)×104 J/m3 and the pre-exponential frequency factor f 0=(4.52±2.93)×1011 Hz. These values are useful in understanding the magnetic behavior of the antiferromagnetic nanoparticles and their potential application in biomedical technology.