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Objective To investigate the effect of fullerol on the osteogenic differentiation of rat adipose-derived mesenchymal stem cells (rADSCs) based on hanging drop culture. Methods rADSC spheres were formed by hanging drop culture for 3 days, then the spheres were dissociated to single cell by trypsin (called rADSC sphere-derived cells). The rADSC sphere-derived cells were cultured in 2-D adherent cell cultures for 24 hours, then the ordinary culture medium was replaced by osteogenic induction medium (osteogenic medium, OM). OM without fullerol served as control group (CM); OM with 1.0μmol/L fullerol was used as the experimental group (OM+Ful1.0μmol/L). Two groups were induced to differentiation for 14d and 21d, using two methods of alizarin red staining and quantitative real-time PCR (qPCR) to detecte the ability of rADSC sphere-derived cells differentiating into osteoblasts. Results rADSCs could form a uniform size microspheres structure through hanging drop culture for 3 days; rADSC sphere-derived cells were obtained by dissipation of trypsin. Compared with OM group, OM+Ful1.0μmol/L can make rADSC sphere-derived cells form more mineralized calcium nodules, and enhance the expression of the relevant osteogenic gene Runx2, OCN and ColI. Conclusion Fullerol can significantly enhance the osteogenic differentiation of rADSCs based on hanging drop culture, and it is helpful to improve the efficiency of osteogenic induction of rADSCs. DOI: 10.11855/j.issn.0577-7402.2016.08.03