Fe3O4 nanoparticles were loaded into poly-L-lactide (PLLA) with concentrations of 2% and 5%, respectively, using an electrospinning method. In vivo animal experiments were then performed to evaluate the potential of the Fe3O4/PLLA nanofibrous material for bone tissue engineering applications. Bony defects with a diameter of 4 mm were prepared in rabbit tibias. Fe3O4/PLLA nanofibers were grafted into the drilled defects and histological examination and computed tomography (CT) image detection were performed after an eight-week healing period. The histological results showed that the artificial bony defects grafted with Fe3O4/PLLA nanofibers exhibited a visibly higher bone healing activity than those grafted with neat PLLA. In addition, the quantitative results from CT images revealed that the bony defects grafted with 2% and 5% Fe3O4/PLLA nanofibers, respectively, showed 1.9- and 2.3-fold increases in bone volume compared to the control blank sample. Overall, the results suggest that the Fe3O4/PLLA nanofibers fabricated in this study may serve as a useful biomaterial for future bone tissue engineering applications.