Spinel ferrites have drawn intensive attention because of their adjustable magnetic properties by ion doping, among which calcium (Ca) is an essential dopant that is widely employed in massive production. However, its exact lattice occupation and relationship with intrinsic magnetic properties remain unclear. Here, we successfully prepared Ca-doped cobalt ferrite (CoFe₂O₄) nanoparticles by electrospinning. Ca²⁺ is observed to occupy both the tetrahedral Fe site and the octahedral Co site using spherical aberration correction transmission electron microscopy (TEM) and prefers to occupy the octahedral site at a high doping level. Such dual occupation behavior affects the tetrahedral and octahedral sublattices differently, resulting in nonmonotonic saturation magnetization variation, reduced magnetocrystalline anisotropy and negative magnetization in the zero field cooling (ZFC) process. By controlling the Ca doping amount, increased saturation magnetization and reduced coercivity can be obtained simultaneously. Our findings establish the relationship between the atomic-scale structural change and the macroscopic magnetic properties of spinel ferrites, promoting the development of new ferrite materials.