Background <p>The association between cholecystectomy and ischemic stroke remains unclear. Therefore, this study aimed to investigate the potential association of genetically predicted liability to cholecystectomy with ischemic stroke and its subtypes using Mendelian randomization (MR).</p> Methods <p>The inverse variance weighted (IVW) method was used as the primary analytical method, with weighted median and MR-Egger regression as complementary methods. Overall ischemic stroke was defined as the primary outcome, whereas ischemic stroke subtypes were considered secondary outcomes. A Bonferroni-corrected significance threshold was applied to the three secondary outcomes. Heterogeneity, horizontal pleiotropy, and the influence of individual variants were assessed using sensitivity analyses.</p> Results <p>Genetically predicted liability to cholecystectomy was not associated with overall ischemic stroke in the primary IVW analysis (OR = 0.57, 95% CI 0.23–1.37, <i>P</i> = 0.208). In the secondary analyses, the inverse association with small-vessel ischemic stroke remained significant after Bonferroni correction (OR = 0.10, 95% CI 0.02–0.57, <i>P</i> = 0.009). The inverse association with large-artery atherosclerotic ischemic stroke was nominally significant but did not meet the corrected threshold (OR = 0.09, 95% CI 0.01–0.71, <i>P</i> = 0.023), whereas no association was observed for cardioembolic ischemic stroke.</p> Conclusion <p>Genetically predicted liability to cholecystectomy was not significantly associated with overall ischemic stroke. The secondary analyses supported an inverse association with small-vessel ischemic stroke and suggested a possible inverse association with large-artery atherosclerotic ischemic stroke. No significant association was observed for cardioembolic ischemic stroke.</p>