Nano-selenium has a great potential to be used in chemical, biological, medical and environmental fields. Biological methods for nano-selenium synthesis have attracted wide interests, because they can be operated at ambient temperature and pressure without complicated equipments. In this work, a protozoa, Tetrahymena thermophila (T. thermophila) SB210, was used to in vivo synthesize nano-selenium. The biosynthesized nano-selenium was characterized using transmission electron microscopy, energy dispersive X-ray spectroscopy and Raman spectroscopy. The synthesized amorphous spherical selenium nanoparticles had diameters of 50-500 nm with the coexistence of irregular nano-selenium. The expressions of glutathione (GSH) synthesis related gene glutathione synthase, cysteine-rich protein metallothionein related gene metallothionein-1 and [2Fe-2S] cluster-binding protein related gene were up-regulated in the nano-selenium producing group. Also, the subsequent GSH detection and in vitro synthesis experimental results suggest the three proteins were likely to be involved in the nano-selenium synthesis process. (C) 2016 Elsevier Inc. All rights reserved.