Hydroxyapatite nanoparticles (HAPN) have been discovered to exert cytotoxicity and apoptosis-induction in some cancer cells. But it is still not clear how tumor cells interact with HAPNs with various sizes. In this study, we investigated the effect of the particle size of the HAPN on the anti-tumor activity, apoptosis-induction and the levels of the apoptotic signaling proteins in human hepatoma HepG2 model cells. HAPNs within 20-180 nm size range were synthesized by a modified sol-gel method. The cellular internalization and biolocalization of the FITC-labeled HAPNs were also identified. The results showed that in HepG2 cells, the anti-tumor activity and HAPN-induced apoptosis strongly depended on the size of HAPNs, and the efficacies all decreased in the order of 45-nm>26-nm>78-nm>175-nm. HAPNs, ranging from 20 nm to 80 nm, were found to effectively activate caspase-3 and -9, decrease the Bcl-2 protein level, and increase the levels of Bax, Bid and the release of cytochrome c from mitochondria into cytoplasm, with the best efficiency from 45-nm HAPN. Correlating the cellular response with the cellular internalization, it can be inferred that the size of HAPN and thereby the cellular localization had predominant effect on the HAPN-induced cytotoxicity, apoptotis, and the levels of the apoptotic proteins in HepG2 cells. The findings presented here could provide new means to modulate the cellular behaviors of HAPN and to guide the design of HAPN-based delivery and therapeutic systems.