Titanium dioxide (titania) nanoparticle aggregation is an important factor in understanding cytotoxicity. However, the effect of the aggregate size of nanoparticles on cells is unclear. We prepared two sizes of titania aggregate particles and investigated their biological activity by analyzing biomarker expression based on mRNA expression analysis. The aggregate particle sizes of small and large aggregated titania were 166 nm (PDI = 0.291) and 596 nm (PDI = 0.417), respectively. These two size groups were separated by centrifugation from the same initial nanoparticle sample. We analyzed the gene expression of biomarkers focused on stress, inflammation, and cytotoxicity. Large titania aggregates show a larger effect on cell viability and gene expression when compared with the small aggregates. This suggests that particle aggregate size is related to cellular effects.