Fullerenols have wide application in the field of life sciences as a result of their extensive biocompatibility and biofunctionality. However, their environmental fate and ecotoxicological risks are largely unknown. In this study, stable isotope (13C) labeling was applied to investigate the bioaccumulation and depuration of fullerenols in Daphnia magna. By incorporation of 13C on the skeleton of fullerenols, the concentrations of fullerenols in the samples could be precisely determined on the basis of carbon isotope ratios (13C/12C). After exposure to 13C-labeled fullerenols in artificial freshwater for 48 h, the steady concentrations of fullerenols in D. magna were nearly 0.39 and 1.37% of the dry body weight in the 0.1 and 1.0 mg/L exposure groups, respectively. After 48 h of depuration, D. magna could excrete 97.34 and 89.56% of the accumulated fullerenols in 0.1 and 1.0 mg/L exposure group, respectively. The depuration of fullerenols from D. magna followed first-order kinetics. Moreover, accumulated fullerenols in gravid D. magna could be transferred to the next generation of neonates. The results in present study demonstrated that stable isotope (13C) labeling is a powerful tool to investigate the environmental fate and the potential impacts of fullerenols in ecological systems.