Objective: To evaluate the safety and efficacy of recombinant human prourokinase (rhPro-UK) administered via intravenous (IV) and intra-arterial (IA) routes in acute ischemic stroke (AIS) patients compared with standard treatments. Methods: A comprehensive search was conducted in accordance with PRISMA guidelines across Scopus, Web of Science, and PubMed until 11 December 2024. Randomized controlled trials (RCTs) assessing rhPro-UK’s efficacy and safety were included. Outcomes included the modified Rankin Scale (mRS), the National Institutes of Health Stroke Scale (NIHSS), mortality, and adverse events (AEs). Data analysis used risk difference (RD) with 95% confidence intervals (CIs). Results: Six RCTs (n = 3993) met the inclusion criteria. IV rhPro-UK showed comparable efficacy to the comparator for the mRS 0–1 at 90 days (RD: 0.00, 95% CI: [−0.04, 0.04]) and the mRS 0–2 (RD: −0.01, 95% CI: [−0.03, 0.01], P = 0.23). IA rhPro-UK significantly improved the mRS 0–1 (RD: 0.13, 95% CI: [0.01, 0.26], P = 0.04). The NIHSS reduction was significant for IV rhPro-UK (MD: −0.83, 95% [CI: −1.36, −0.29]). IV rhPro-UK did not significantly reduce the risk of systemic bleeding (RD: −0.10, 95% CI: [−0.24, 0.03], P = 0.12), serious AEs (RD: −0.01, 95% CI: [−0.04, 0.02], P = 0.53), or mortality (RD: 0.01, 95% CI: −0.01, 0.02). IA rhPro-UK significantly increased hemorrhage with neurological deterioration (RD: 0.08, 95% CI: [0.01, 0.14], P = 0.02). Conclusions: IV rhPro-UK provides non-inferior efficacy to both alteplase and standard care with a better safety profile at 35 mg, while IA rhPro-UK enhances functional outcomes in middle cerebral artery occlusions, albeit with safety concerns. Further trials are needed to confirm long-term outcomes, optimal dosing, and broader applicability.