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As lanthanide-doped sodium yttrium flouride (NaYF4) nanoparticles have great potential in biomedical applications, their biosafety is important and has attracted significant attention. In the present work, three different sized NaYF4:Eu3+nanoparticles have been prepared. Liver BRL 3A cell was used as a cell model to evaluate their biological effects. Cell viability and apoptosis assays were used to confirm the cytotoxicity induced by NaYF4:Eu3+NPs. Apart from the elevated malondialdehyde (MDA), the decrease of superoxide dismutase (SOD), glutathione peroxidase (GSH-PX) and catalase (CAT) activity indicated reactive oxygen species (ROS) generation, which were associated with oxidative damage. The decrease of mitochondrial membrane potential (MMP) value demonstrated the occurrence of mitochondria damage. Then, release of cytochrome c from mitochondria and activation of caspase-3 confirmed that NaYF4:Eu3+NPs induced apoptosis was mitochondria damage-dependent.