Fe3O4/carbon (Fe3O4/C) composite microspheres with a high content of Fe3O4 nanoparticles as an active material are prepared by suspension polymerization and heat treatment. A significant difference is observed in the morphology of the Fe3O4/C composite microspheres with the introduction of different amounts of Fe3O4 nanoparticles. The morphological and structural differences of the Fe3O4/C composite microspheres are characterized by focused ion beam cross-section, scanning electron microscopy, transmission electron microscopy, and X-ray diffraction analysis. Thermogravimetric analysis is conducted to measure the amount of Fe3O4 nanoparticles introduced into a carbon matrix. We fix two samples with Fe3O4 contents of 72 wt.% and 98 wt.%, respectively. Fe3O4/C composite microspheres containing 72 wt.% Fe3O4 nanoparticles show much higher capacity retention with an excellent columbic efficiency of 99% at every cycle in comparison with that of the Fe3O4/C composite microspheres containing 98 wt.% Fe3O4 nanoparticles when used as anodes for LIBs. These results indicate that the particle morphology and weight ratio of incorporated Fe3O4 to carbon matrix affects the electrochemical performance of Li-ion cells.