Objective: To prepare oligomeric forms of α-synuclein (α-Syn) in vitro and investigate the effects of α-Syn oligomers on mitochondrial membrane potential of dopaminergic neural cells. Methods: Recombinant human α-Syn in phosphate-buffered saline (PBS) was incubated with continuous shaking. Western blotting analysis and double-antibody sandwich ELISA were used to examine the formation rule of α-Syn oligomers. Immunofluorescent double labeling was used to observe the transporting and distribution of α-Syn monomers and oligomers in vitro culture of MES23.5 dopaminergic neural cells. Mitochondrial membrane potential in the protein-treated cells was assayed by rhodamine method. Results: Different forms of α-Syn oligomers were formed after incubation in PBS, which include dimers, trimers, tetramers and 14-polymers. As shown by ELISA, the quantity of α-Syn oligomers increased with the prolongation of the incubation time and the enhancement of the monomers concentration (P < 0.01, for all). When applied to culture medium, both the monomers and the oligomers could be transported into the cells, where they were further transported onto mitochondria. Both the monomers and the oligomers reduced the mitochondrial membrane potential, the differences with control group were both significant (P = 0.000, for all). However, the oligomers showed much more significant reducing effect on the mitochondrial membrane potential than the monomers (P = 0.000). Conclusion: α-synuclein can form different forms of oligomers by incubation in vitro, α-synuclein oligomers can enter dopaminergic neural cells and obviously reduce the mitochondrial membrane potential.