To investigate the protective effect of miR-218-5p on dopaminergic neurons in the substantia nigra compacta (SNc) by establishing a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mouse model of Parkinson’s disease (PD). Methods: A total of 18 8-week-old male C57 mice were randomly divided into MPTP group and PBS group. Mice were injected intraperitoneally with MPTP (20 mg/kg) or with an equal amount of PBS every 2 h for a total of 4 doses. An additional 36 8-week-old adult male C57 mice were randomly divided into 4 groups with 9 mice in each: (NC agomir+PBS) (NCPBS) group, (NC agomir+MPTP) (NCMPTP) group, (miR-218-5p agomir+PBS) (218PBS) group, and (miR-218-5p agomir+ MPTP) (218MPTP) group. Bilateral stereotaxic injections of miR-218-5p agomir or NC agomir in the SNc were performed respectively, then 3 days later, mice were injected intraperitoneally with MPTP or PBS as before. The expression of tyrosine hydroxylase (TH) protein in the SNc was detected by immunofluorescence and Western blot (WB). The expression of miR-218-5p, Wnt7a mRNA, Ctnnb1 mRNA, Lef1 mRNA, and Birc5 mRNA in the SNc was assessed with quantitative real-time PCR (qPCR). Results: Compared with the PBS group, the expression of miR-218-5p of SNc in the MPTP group decreased obviously (P<0.05). Compared with NCPBS group mice, the NCMPTP group mice showed a reduced number of TH+ cells, decreased expression of TH protein, and decreased expression of Wnt7a, Ctnnb1, Lef1, and Birc5 mRNA in the SNc (P<0.05). Compared with NCMPTP group mice, the 218MPTP group mice had a greater number of TH + cells, increased expression of TH protein, and increased expression of Wnt7a, Ctnnb1, Lef1, and Birc5 mRNA (P<0.05). Conclusion: MiR-218-5p significantly alleviates MPTP-induced dopaminergic neuronal degeneration in the SNc, potentially via Wnt/ β-catenin signaling. This finding may provide a potential therapeutic target for the treatment of PD.