Recombinant prourokinase (rhPro-UK) is a specific plasmin activator, which has been approved to treat acute myocardial infarction in China. This phase 3 trial aimed to further demonstrate the efficacy and safety of rhPro-UK in patients with acute ischemic stroke (AIS) within 4.5 h of symptom onset. RhPro-UK in AIS within 4.5 h of stroke onset trial-2 (PROST-2) is a multicenter, prospective randomized, open-label, blinded end-point, non-inferiority, recombinant tissue plasmin activator (rt-PA)-controlled, phase 3 trial. A total of 1552 patients who are eligible for intravenous thrombolytic therapy from 72 clinical sites will be randomly assigned to receive either rhPro-UK 35 mg (15 mg bolus + 20 mg infusion/30 min) or rt-PA 0.9 mg/kg (10% bolus + 90% infusion/1 h). The primary outcome is the proportion of patients with a modified Rankin Scale (mRS) score of 0-1 at 90 days. Secondary efficacy outcomes include the proportion of patients with mRS score of 0-2, the distribution of mRS, self-care ability in daily life on the Barthel Index at 90 days, the proportion of subjects with ⩾ 4 points decrease in National Institutes of Health Stroke Scale (NIHSS) score or NIHSS score ⩽ 1 from baseline at 24 h and 7 days after treatment. Safety outcomes are symptomatic intracranial hemorrhage (sICH) and major systematic bleeding within 7 days as well as death from all causes within 90 days. The results from the PROST-2 trial will comprehensively elucidate the efficacy and safety profile of rhPro-UK as a potential alternative agent for stroke thrombolysis. URL: http://www.clinicaltrials.gov. Unique identifier: NCT05700591.