BACKGROUND: Recent research has indicated a possible link between the use of methotrexate (MTX) and a heightened risk of developing Parkinson's disease (PD). Nevertheless, the causal relationship between MTX and PD continues to be unclear. This study aimed to explore the potential causal impact of MTX use on the risk of PD by employing two-sample Mendelian randomization (MR) alongside co-localization (COLOC) analysis. OBJECTIVE: The objective of this research is to explore the potential causal relationship between the use of methotrexate and the likelihood of developing Parkinson's disease, employing a two-sample Mendelian randomization (TSMR) methodology. METHODS: Separate datasets concerning the genetic tools associated with MTX and PD were acquired from the Genome-Wide Association Study (GWAS) database. A series of MR-related statistical analyses were executed, such as inverse variance weighting (IVW), weighted median (WM 1), weighted mode (WM 2), and MR-Egger regression techniques. Furthermore, we carried out co-localization analyses utilizing the GWAS summary statistics for both MTX and PD in order to comprehensively evaluate the causal relationship between MTX and the risk of developing PD. RESULTS: The MR analysis revealed a positive causal connection between methotrexate (MTX) and a decreased likelihood of developing Parkinson's disease (PD). In particular, the IVW method showed a negative association between MTX use and PD incidence, indicating that MTX is linked to a lower risk of PD (OR = 4.78E-11, 95 % CI = 1.06E-19 to 2.16E-02, p = 1.94E-08). Similar findings were acquired through the WM 1, WM 2, and MR-Egger methodologies. Additionally, COLOC analysis indicated a shared genetic variant between MTX and PD at a specific locus. CONCLUSION: The results from this joint MR and COLOC research indicate a possible causal link between the use of methotrexate and the likelihood of developing Parkinson's disease. Nonetheless, further research and confirmation of these results, as well as an examination of potential mechanisms, are necessary.