The current European Stroke Organisation expedited recommendation on tenecteplase (TNK) for acute ischemic stroke (AIS) advocates that TNK 0.25 mg/kg can be used alternatively to alteplase (tissue plasminogen activator [TPA]) for AIS of <4.5 hours duration, based on a meta-analytical approach establishing noninferiority. Since the publication of these guidelines, 4 additional randomized controlled clinical trials (RCTs) have provided further insight. We conducted an updated systematic review and meta-analysis including all available RCTs that investigated efficacy and safety of TNK 0.25 mg/kg compared with TPA for the treatment of AIS within 4.5 hours of onset. The primary outcome was defined as the excellent functional outcome at 3 months (modified Rankin Scale [mRS] score 0-1), whereas good functional outcome (mRS score 0-2), reduced disability at 3 months (≥1-point reduction across all mRS scores), symptomatic intracranial hemorrhage (sICH), and 3-month mortality were evaluated as secondary outcomes. Pooled estimates were calculated with random-effects model. A prespecified subgroup analysis was performed stratifying for TNK formulation, that is, original TNK vs biocopy: recombinant human TNK tissue-type plasminogen activator that is available in China and has a different production process. Eleven RCTs were included comprising a total of 3,788 patients treated with TNK vs 3,757 patients treated with TPA. TNK was associated with higher likelihood of excellent functional outcome (risk ratio [RR] 1.05, 95% CI 1.01-1.10; p = 0.012; I2 = 0%; risk difference 2.95%; 95% CI 0.76%-5.14%; p = 0.008; I2 = 0%) and reduced disability at 3 months (common odds ratio 1.10, 95% CI 1.01-1.19; p = 0.034; I2 = 0%) compared with TPA while good functional outcome (RR 1.03, 95% CI 0.99-1.07; p = 0.142; I2 = 28%) was similar between the groups. Regarding safety outcomes, similar rates of sICH (RR 1.12, 95% CI 0.83-1.53; p = 0.456; I2 = 0%) and 3-month mortality (RR 0.97, 95% CI 0.82-1.15; p = 0.727; I2 = 12%) were observed. When stratified for TNK regimen (original vs biocopy), statistical significance in achieving an excellent functional outcome at 3 months was retained for the original TNK (RR 1.05, 95% CI 1.00-1.10; p = 0.044; I2 = 0%). The updated meta-analysis confirms similar safety between TNK 0.25 mg/kg and TPA, while showing that TNK is superior to TPA regarding excellent functional outcome and reduced disability at 3 months. These findings support transitioning to TNK in clinical practice.