Our previous research revealed that rifampicin could protect PC12 (pheochromocytoma 12) cells from rotenone-induced cytotoxicity by reversing the aggregation of alpha-synuclein. Furthermore, increasing evidence indicated that the misfolded alpha-synuclein with SUMOylation, an important protein post translational modification, was easier to solubilize and was less toxic. Here, we investigated whether rifampicin could stabilize alpha-synuclein and prevent rotenone-induced PC12 cells from undergoing apoptosis by enhancing SUMOylation of alpha-synuclein. The expression of SUMO1 and SUMO2/3, the two main proteins responsible for the SUMOylation modification in PC12 cells, were detected by western blotting. Co-immunoprecipitation was performed to compare qualitatively the SUMOylation modification of alpha-synuclein. The cell viability and apoptosis rate were measured by a CCK-8 assay kit and flow cytometry, respectively. We targeted Ubc9 as a key enzyme in the SUMOylation modification pathway and knocked down the UBC9 gene using a short interfering RNA. Treatment with 150 mu mol/L rifampicin, increased the expressions of SUMO1 and SUMO2/3 in cells by 1.5 times compared with the control group; meanwhile, the cell viability of rotenone-induced cells increased from 20 to 80% (P < 0.05). In addition, the increased SUMOylation activity in the cells stimulated by rifampicin was observed 18 h earlier compared with cells treated by rotenone alone. SUMOylation of alpha-synuclein was more significant in rifampicin-treated cells and Ubc9 upregulated cells. However, the same phenomenon and the protective effect of rifampicin were reversed after UBC9 knockout. In conclusion, rifampicin might reduce the cytotoxicity of rotenone-induced PC12 cells by promoting SUMOylation of cc-synuclein. (C) 2017 Elsevier Inc. All rights reserved.