Previous studies evaluating the association between antidiabetic drugs and the risk of ischemic stroke have yielded inconsistent results. This study employed a Mendelian randomization (MR) approach to preliminarily explore the potential causal associations of major antidiabetic drugs with the risk of ischemic stroke and its subtypes, and performed validation using independent European and Asian population data. Methods: Genetic variants associated with the targets of sulfonylureas (ABCC8/KCNJ11), GLP-1 analogs (GLP1R), thiazolidinediones (PPARG), insulin and its analogs (INSR), and metformin were extracted from published studies as instrumental variables (all instrument F-statistics>10). The primary analysis was conducted using MEGASTROKE European population data. Inverse variance weighting (IVW) or the Wald ratio method was used to estimate the causal effects of drug targets on acute ischemic stroke (AIS), large artery atherosclerosis stroke (LAS), cardioembolic stroke (CES), and small vessel stroke (SVS). External validation consisted of: (1) validation of overall AIS using independent European population data from FinnGen; (2) an exploratory analysis of stroke subtypes using GIGASTROKE East Asian population data. This study was exploratory in nature; no corrections were made for multiple comparisons, and a P-value < 0.05 was used as the statistical criterion for nominal association. Results: The primary analysis (MEGASTROKE) showed that sulfonylureas (rs757110) had a nominal association with reduced AIS risk (OR=0.05, 95% CI 0.01-0.29, P=0.001), although the effect was extreme; metformin showed a suggestive association with LAS (OR=0.47, P=0.047); and the multi-single nucleotide polymorphism (SNP) instrument for sulfonylureas (KCNJ11/ABCC8) showed a suggestive association with CES (OR=0.09, P=0.046). External validation: In FinnGen, none of the associations between the drugs and AIS were statistically significant (rs757110: OR=0.53, P=0.601; all others P>0.05). In the exploratory analysis of the East Asian population, none of the original positive signals for stroke subtypes showed a nominal association [metformin with LAS: OR=1.22, P=0.715; sulfonylureas (ABCC8/KCNJ11) with CES: OR=1.68, P= 0.871]; the associations were not replicated, and the confidence intervals were extremely wide, suggesting insufficient statistical power. Conclusion: The positive signals for antidiabetic drugs on ischemic stroke observed in the primary analysis were not supported by independent external validation. The extreme protective effect of the sulfonylurea SNP-rs757110 was judged as a false positive. The suggestive associations of metformin with LAS and sulfonylureas with CES were not replicated in external validation and should be considered only as exploratory hypotheses. The current genetic evidence does not support the clinical application of any antidiabetic drug for ischemic stroke or its subtypes.