Lanthanide upconversion nanophosphors (UCNPs) show unique upconversion luminescence where lower-energy photons (such as near-infrared (NIR) excitation) are converted into higher-energy photons covering the NIR to the UV region, and are considered to have a bright future in clinical translation. As UCNPs are used in a significant number of potential bio-applications, their biosafety is important and has attracted significant attention. In this critical review, recent reports regarding the cellular internalization, biodistribution, excretion, cytotoxicity and in vivo toxic effects of UCNPs are reviewed. In particular, the studies which evaluated the association between the chemical and physical properties of UCNPs and their biodistribution, excretion, and toxic effects are presented in detail. Finally, we also discuss the challenges of ensuring the biosafety of UCNPs in vivo.