Neuro-cytotoxicity of iron oxide nanoparticles (NPs) need to be considered. In this paper, magnetic Fe3O4 NPs are synthesized via a bio-mineralization process using silk fibroin (SF) as template. The prepared NPs with SF (SF-NPs) have more uniform grain size, better stability in weakly alkaline solution and higher saturation magnetization (about 82 emu/g) than pure NPs. After exposure to different concentration of SF-NPs (6.25-100 μg/mL), the reactive oxygen species generation in PC12 cells reduced compared with pure NPs. 1-5d treatment with SF-NPs did not destroy cell membrane and cyto-skeleton, and could improve the neurites extension in a dose-dependent manner at lower concentration (6.25-50 μg/mL), because SF peptide coating could delay the release of iron ions and the increase of surface crystal defects of NPs. Intact mitochondria in a neurite indicate the extension activity of neurites of cells treated with SF-NPs. Their high magnetic property and good neural-cytocompatibility provide the possibility of SF-NPs to be used as the contrast agents in brain MRI or the carrier of neural therapeutic drugs.