Objective To construct a lentiviral vector for RNA interference of the Sirt3 gene and to establish a Sirt3 knockdown human neuroblastoma SH-SY5Y cell line. Methods According to the Sirt3 nucleotide sequence archived in the GenBank database, four siRNAs targeting Sirt3 and one negative control were designed, and cloned into a linear vector containing the green fluorescent protein ( GFP ) gene to produce four recombinant lentivirus plasmids. The recombinant plasmids and helper plasmids were transfected into 293T cells, and the titer of the virus was determined. SH- SY5Y cells were infected with the constructed lentivirus, and the silencing effect on Sirt3 was accessed by real-time PCR and Western blot. The lentivirus-infected cells were screened for the most significant Sirt3 knockdown. Results Recombinant lentiviral vectors expressing siRNAs targeting Sirt3 were successfully constructed and confirmed by DNA sequencing. The viral titers of the recombinant lentivirus were as follows: LV-Sirt3-RNAi-1 8×10⁸ TU/ mL, LV-Sirt3- RNAi-2 3×10⁸ TU/ mL, LV-Sirt3-RNAi-3 8× 10⁸ TU/ mL, and LV-Sirt3-RNAi-4 8× 10⁸ TU/ mL. The levels of Sirt3 mRNA and Sirt3 protein in the LV-Sirt3-RNAi-3 group were significantly less than those in the negative control group and the blank control group ( P < 0. 001, P < 0. 001). Conclusions A lentivirus vector for RNA interference of Sirt3 was successfully constructed and SH-SY5Y cell lines with Sirt3 gene knockdown were selected, which will be useful for future research of Sirt3 function in Parkinson’s disease cell models.