OBJECTIVE: We evaluated the effect of two doses of natalizumab on functional outcomes in acute ischemic stroke (AIS) patients. METHODS: In this double-blind phase 2b trial, AIS patients aged 18-80 years with National Institutes of Health Stroke Scale scores of 5-23 from 53 US and European sites were randomized 1:1:1 to receive a single dose of 300 or 600 mg intravenous natalizumab or placebo, with randomization stratified by treatment window (≤9 or >9 to ≤24 hours from patient's last known normal state). The primary endpoint was a composite measure of excellent outcome (modified Rankin Scale score ≤1 and Barthel Index score ≥95) at day 90 assessed in all patients receiving a full dose. Sample size was estimated from a Bayesian model; p values were not used for hypothesis testing. RESULTS: An excellent outcome was less likely with natalizumab than with placebo (natalizumab 300 mg or 600 mg odds ratio 0.60; 95% confidence interval 0.39-0.93). There was no effect modification by time to treatment or use of thrombolysis/thrombectomy. For natalizumab 300 mg, 600 mg, or placebo, there were no differences in incidence of adverse events (90%, 92%, and 92%, respectively), serious adverse events (26%, 33%, and 21%, respectively), or deaths (7%, 5%, and 6%, respectively). CONCLUSIONS: Natalizumab administered ≤24 hours after AIS did not improve patient outcomes. CLINICALTRIALSGOV IDENTIFIER: NCT02730455 CLASSIFICATION OF EVIDENCE: This study provides Class I evidence that for patients with AIS, an excellent outcome was less likely in patients treated with natalizumab than with placebo.