Parkinson's disease (PD) is the world's second most prevalent neurodegenerative disease. Currently, aside from levodopa, there are no other effective drugs clinically available to slow its progression. Otx2 plays a critical role in the differentiation of midbrain dopaminergic neurons (mDANs) during midbrain development. However, in adulthood, Otx2 is primarily expressed in the ventral tegmental area (VTA)-ventral part, and mDANs in the dorsal part of the VTA and the substantia nigra pars compacta (SNc) show no Otx2 expression. Research indicates that Otx2 is essential not only for the development of mDANs but also for their protection against the toxicity of MPTP and rotenone. Consequently, Otx2 is a potential clinical target for mDANs protection. Identifying the upstream mechanism that regulates Otx2 expression is crucial to restoring its expression in the SNc and enhancing its levels in the entire ventral midbrain mDANs. In this study, we have demonstrated the safety of Otx2 overexpression in vitro by using adeno-associate virus (AAV) and explored the feasibility of promoting Otx2 expression through the Wnt/β-Catenin signaling pathway using various drugs, a miR-34 mimic, and an inhibitor. Our results showed that Otx2 overexpression via AAV in the SNc is relatively safe, and CHIR99021 can induce Otx2 expression in mouse mDANs, thereby, alleviating PD-liked motor symptoms induced by MPTP. These findings suggest that modulating Otx2 expression through the Wnt/β-Catenin signaling pathway holds a therapeutic approach for Parkinson's disease.