No reports regarding the safety of thrombolysis in acute stroke patients with a G6PD deficiency have been published to date. Here we aimed to evaluate the safety of intravenous thrombolysis for G6PD-deficient stroke patients. We enrolled each patient with acute ischemic stroke who arrived in our stroke unit within the therapeutic window and received systemic thrombolysis using recombinant tissue plasminogen activator (rt-PA), between January 2015 and March 2016. The primary clinical outcome was measured 3 months after treatment, and defined as a "good" outcome by a modified Rankin Scale (mRS) score of 0-2. Major safety outcomes were incidences of intracranial hemorrhage (ICH) or mortality at 90 days. A total of 96 individuals were analyzed, of which 20 patients were G6PD deficient. The rates of ICH after rt-PA treatment were 12% the in G6PD-deficient group versus 15% in G6PD non-deficient group, and the incidences of symptomatic intracranial hemorrhage were also similar between the G6PD-deficient and non-deficient cohorts. No hemolysis crisis occurred, and no significant difference in mortality rate was found between the 2 groups. The overall rate of a good outcome at 3 months after stroke in the whole cohort was 60%, whereas 50% of patients achieved an excellent outcome (mRS 0-1) in the G6PD-deficient cohort, and 42% in the G6PD non-deficient group. Thrombolytic therapy for patients with G6PD deficiency seems to pose a similar risk of ICH and clinical outcome to those with G6PD non-deficiency.