Abstract The physicochemical properties of fullerene water suspensions (nC 60) and their subsequent toxicity were influenced by different preparation methods. The nC 60 suspensions were produced by three methods: toluene exchange (Tol/nC 60), DMSO dissolving (DMSO/nC 60), and stirring overtime (Aqu/nC 60). The particle size, zeta potential, and nC 60 structure were strongly dependent on both the type of aggregates formed and the test medium addition. Specifically, Tol/nC 60 exhibited small and spherical closed aggregates, whereas DMSO/nC 60 and Aqu/nC 60 presented mesoscale aggregates of smaller spherical aggregates. These differences in the physicochemical properties of nC 60 determined the embryonic toxicity and oxidative stress of Japanese medaka ( Oryzias latipes). The mortality and glutathione (GSH) induction of embryos were ranked in the order of Tol/nC 60 > DMSO/nC 60 > Aqu/nC 60, and the morphological malformations were in the order of DMSO/nC 60 > Tol/nC 60 > Aqu/nC 60. The mortality of Tol/nC 60 was attributed to its closely packed fullerene structure, which remained as largely underivatized C 60. The malformations of DMSO/nC 60 might have originated from the co-effect of organic solvent remaining in the fullerene colloid. To summarize, these findings clearly illustrated the need to consider the effect of preparation method on the physicochemical properties when assessing nC 60 toxicity.