Objective：To analyze the effects of bufalin on autophagy induced by gentamicin(GM) in rat renal tubular epithelial cells (NRK52e) in vitro. Methods：NRK52e cells were cultured in vitro and divided into 4 groups: the control group, the GM group (2 mg/mL), the bufalin group(1×10-8 mol/L) and the GM+bufalin group(GM 2 mg/mL+bufalin 1×10-8 mol/L). The autophagy of NRK52e cells was observed by transmission electron microscopy. Effects of GM and bufalin on the expression of autophagy-related protein LC3Ⅱ and P62, and kidney injury molecule-1(KIM1) protein were examined by Western blot. Results：Transmission electron microscopy showed that the number of autophagosomes in the GM group was significantly increased compared with the control group, and the number of autophagosomes of the bufalin group was less than that in the GM group. Western blot showed that the relative expressions of LC3Ⅱ, P62 and KIM1 protein were significantly increased in the GM group compared with the control group (all P<0.05), however, the relative expressions of LC3Ⅱ, P62 and KIM1 protein were decreased in the bufalin group than those in the GM group (P<0.05). Conclusion：Gentamicin could activate the autophagy of NRK52e cells and up-regulate the expression of KIM1 protein, while bufalin could partially inhibit GM-induced NRK52e cell’s autophagy and the up-regulated expression of KIM1 protein. It may be the mechanisms by which bufalin acts the renoprotection.