Background: Outcome data regarding the administration of tenecteplase (TNK) to acute ischemic stroke (AIS) patients presenting in the extended time window are limited. Objectives: We aimed to assess the current evidence regarding the efficacy and safety of TNK at a dose of 0.25 mg/kg for AIS treatment in the extended time window. Design: A systematic review and meta-analysis was conducted including all available randomized-controlled clinical trials (RCTs) that compared TNK 0.25 mg/kg versus no thrombolysis in AIS patients presenting in the extended time window (>4.5 h after last-seen-well or witnessed onset). Data sources and methods: Eligible studies were identified by searching Medline, Scopus, and international conference abstracts. The predefined efficacy outcomes of interest were 3-month excellent functional outcome [defined as the modified Rankin Scale (mRS) score ⩽1; primary outcome], 3-month good functional outcome (mRS ⩽ 2), 3-month reduced disability (⩾1-point reduction across all mRS scores). We determined symptomatic intracranial hemorrhage (sICH), any ICH and 3-month mortality as safety endpoints. A random-effects model was used to calculate risk ratios (RRs) and common odds ratios (cORs) with corresponding 95% confidence intervals (CIs). Results: Three RCTs were included comprising 556 patients treated with TNK versus 560 controls. TNK 0.25 mg/kg was associated with a higher likelihood of 3-month excellent functional outcome compared to controls (RR = 1.17; 95% CI = 1.01–1.36; I² = 0%), whereas there was no difference regarding good functional outcome (RR = 1.05; 95% CI = 0.94–1.17; I² = 0%) and reduced disability (adjusted cOR = 1.14; 95% CI = 0.92–1.40; I² = 0%) at 3 months. The risks of sICH (RR = 1.67; 95% CI = 0.70–4.00; I² = 0%), any ICH (RR = 1.08; 95% CI = 0.90–1.29; I² = 0%) and 3-month mortality (RR = 1.10; 95% CI = 0.81–1.49; I² = 0%) were similar between the groups. Conclusion: Based on data from three RCTs showing increased efficacy and a favorable safety profile of TNK in the treatment of AIS in the extended time window, continuing efforts of ongoing RCTs in the field are clearly supported. Trial registration: PROSPERO registration ID: CRD42023448707.