Silica-titania hollow nanoparticles (HNPs) with uniform diameters of 25, 50, 75, 100, and 125 nm were fabricated by dissolution and redeposition method in order to evaluate size dependent cellular response. Surface-modified HNPs with cationic, anionic, and neutral functional group were prepared by silane treatment. We systematically investigated cellular internalization, toxicity, and innate immune response of HNPs in human breast cancer (SK-BR-3) and mouse alveolar macrophage (J774A.1) cells. Size- and surface functionality-dependent cellular uptake of HNPs was investigated by fluorescence labeling (fluorescein isothiocyanate), inductively coupled plasma-emission spectroscopy, and ultrastructural resolution using transmission electron microscopy. Viability, reactive oxygen species, and apoptosis/necrosis of HNP-treated J774A.1 revealed the size-dependent phenomenon. Innate immune response of HNP-treated macrophages was measured by three cytokines such as interleukin-1, interleukin-6, and tumor necrosis factor α. Among the HNPs of different sizes, 50-nm HNPs demonstrated the highest toxic influence on macrophages. Among the HNPs with surface functionalities, cationic HNPs demonstrated the most toxic effect on J774A.1 and the highest uptake efficiency. The toxicity results of HNP-treated macrophages were consistent with the cellular internalization efficiency. These findings provide size- and surface functionality-dependent nanotoxicity and uptake of HNPs, and lead to HNPs for bioapplications such as drug delivery and imaging probe.