As the most widely application of nanomaterials in biology and medicine, their interaction with biological system and the afterwards cellular responses would be addressed. Here, the agglomerate states of two kinds of TiO2 NPs in culture medium were characterized and the cluster specific cellular responses in RAW264.7 cells were investigated. Owing to the smaller aggregates and more positively charged surface, 21 nm TiO2 NPs exhibited higher cytotoxicity, which correlated with their ability to cause damage to mitochondria. While for 35 nm TiO2 NPs, higher level of cell autophagy and stronger pro-inflammatory immune response were observed, which are responsible for their lower cytotoxicity. These results suggest that physiochemical properties of TiO2 NPs in culture medium are important factor affecting their cellular response to RAW264.7 cells.