Superparamagnetic magnetite nanoparticles are of great interest owing to their numerous existing and potential biomedical applications. In this study, superparamagnetic magnetite nanoparticles with average diameters of 6–8 nm have been prepared and surface-functionalized with poly(N-isopropylacrylamide) (poly(NIPAAM)) via a surface-initiated atom-transfer radical polymerization, followed by immobilization of heparin. The success of the various surface-functionalization steps has been ascertained using Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy. The rate of internalization of the as-synthesized and surface-functionalized magnetite nanoparticles by mouse macrophage cells has been investigated. The nanoparticle internalization into the macrophages has been visualized using optical microscopy and quantified by inductively coupled plasma spectroscopy. The effectiveness of the heparinized nanoparticles in preventing thrombosis has been determined using the plasma recalcification time. The results indicate that the above-mentioned surface modifications of the magnetite nanoparticles are effective in delaying phagocytosis and preventing blood clotting in vitro. Such properties can be expected to enable their use in biomedical applications.