Background: and Purpose We performed a two-sample Mendelian randomization (MR) analysis to evaluate the causal effect of genetically proxied AMP-activated protein kinase (AMPK) activation, which is the target of metformin, on functional outcome following ischemic stroke onset. Methods: A total of 44 AMPK-related variants associated with HbA1c (%) were used as instruments for AMPK activation. The primary outcome was the modified Rankin Scale (mRS) score at 3 months following the onset of ischemic stroke, evaluated as a dichotomous variable (3–6 vs. 0–2) and subsequently as an ordinal variable. Summary-level data for the 3-month mRS were obtained from the Genetics of Ischemic Stroke Functional Outcome network, including 6,165 patients with ischemic stroke. The inverse-variance weighted method was used to obtain causal estimates. The alternative MR methods were used for sensitivity analysis. Results: Genetically predicted AMPK activation was significantly associated with lower odds of poor functional outcome (mRS 3–6 vs. 0–2, odds ratio [OR]: 0.06, 95% confidence interval [CI]: 0.01–0.49, P=0.009). This association was maintained when 3-month mRS was analyzed as an ordinal variable. Similar results were observed in the sensitivity analyses, and there was no evidence of pleiotropy. Conclusion This MR study provided evidence that AMPK activation by metformin may exert beneficial effects on functional outcome following ischemic stroke.