Lepalvir, derived from inflamed rabbit skin inoculated with vaccinia virus, has potential neuroprotective and anti-inflammatory effects. We conducted a phase II, multicenter, randomized, blind, placebo-controlled trial investigating the efficacy and safety of Lepalvir for acute ischemic stroke (AIS). Participants aged 18-80 years with AIS in the anterior circulation and a National Institutes of Health Stroke Scale (NIHSS) score of 4-24 within 48 h post-onset were randomized to receive high-dose (192U), low-dose (96U) Lepalvir, or saline placebo for 14 days. The primary outcome was the proportion of patients achieving a modified Rankin Scale (mRS) score <= 1 at day 90 (D90) post-randomization. Among 238 patients, no significant difference in mRS score at D90 was observed across groups, yet a higher percentage in the high-dose group achieved a mRS score <= 1 at D90, compared to the control and low-dose group. No significant safety concerns were noted. While functional improvement was not significantly different at D90, Lepalvir showed a favorable safety profile and potential at the higher dosage, warranting further phase III investigation.