Highly crystalline single-domain magnetite Fe₃O₄nanoparticles (NPs) are important, not only for fundamental understanding of magnetic behaviour, but also for their considerable potential applications in biomedicine and industry. Fe₃O₄NPs with sizes of 10–300 nm were systematically investigated to reveal the fundamental relationship between the crystal domain structure and the magnetic properties. The examined Fe₃O₄NPs were prepared under well-controlled crystal growth conditions using a large-scale liquid precipitation method. The crystallite size of cube-like NPs estimated from X-ray diffraction pattern increased linearly as the particle size (estimated by transmission electron microscopy) increased from 10 to 64.7 nm, which indicates that the NPs have a single-domain structure. This was further confirmed by the uniform lattice fringes. The critical size of approximately 76 nm was obtained by correlating particle size with both crystallite size and magnetic coercivity; this was reported for the first time in this study. The coercivity of cube-like Fe₃O₄NPs increased to a maximum of 190 Oe at the critical size, which suggests strong exchange interactions during spin alignment. Compared with cube-like NPs, sphere-like NPs have lower magnetic coercivity and remanence values, which is caused by the different orientations of their polycrystalline structure.