Aggregation appears to be a ubiquitous phenomenon among all nanoparticles and its influence in mediating cellular uptake and interactions remain unclear. Here we developed a simple technique to produce transferrin-coated gold nanoparticle aggregates of different sizes and characterized their uptake and toxicity in three different cell lines. While the aggregation did not elicit a unique toxic response, the uptake patterns were different between single and aggregated nanoparticles. There was a 25% decrease in uptake of aggregated nanoparticles with HeLa and A549 cells in comparison to single and monodisperse nanoparticles. However, there was a 2-fold increase in MDA-MB 435 cell uptake for the largest synthesized aggregates. These contrasting results suggest that cell type and the mechanism of interactions may play a significant role. This study highlights the need to investigate the behavior of aggregates with cells on a case-by-case basis and the importance of aggregation in mediating targeting and intracellular trafficking.