The superparamagnetic magnetite nanoparticles were synthesized through co-precipitation method by using a strong base such as sodium hydroxide or a weak base such as ammonium hydroxide. The magnetite co-precipitated by ammonium hydroxide (MA) has different properties than the magnetite co-precipitated by sodium hydroxide (MS). The cytotoxicity effects of MA and MS on the breast cancer cells and normal hepatocytes cells were studied. The magnetite nanoparticles with two ways were characterized by using Vibrating Sample Magnetometer. X-ray fluorescence, Dynamic Light Scattering, Zeta Potential, pH changes, Wide-angle X-ray Diffraction, Fourier Transforms Infrared spectroscopy, MTT assay test and High-Resolution Transmission electron microscopy. The results showed that the final pH of MA and MS were 5 and 7.5, respectively. MA nanoparticles have salts which act as weak oxidizing agent and they were exposed to oxidation at high temperature and lost their magnetic property. They have a cytotoxic effect against breast cancer cells and normal hepatocytes cells more than the MS.