Using magnetic nanoparticles to enhance gene transfection, a recently developed technique termed magnetofection, has been shown to be a powerful technology in gene delivery. The most widely used magnetic nanoparticles in this area are those coated with polyethyleneimine, which is a well known non-viral transfection agent. In this article, we report methods to control the aggregate size of polyethyleneimine-coated magnetite particles. These particles were then used to enhance transfection of green fluorescent protein (GFP) into NIH 3T3 cells in vitro. We find that the aggregate size of the particles has a great effect on their performance in magnetofection, with less aggregated magnetic particles being more effective in enhancing the gene transfection.