Objective To explore the effect of tissue kallikrein 1 (KLK1) on mitochondrial function after cardiac ischemia/reperfusion (I/R) injury and its mechanism. Methods After KLK1 overexpression by KLK1 recombinant adenovirus infection, the cardiac I/R rat model was established by ligation of left anterior descending coronary artery and reperfusion. The cardiac infarction area and the apoptosis of cardiomyocytes were detected. The mitochondria were isolated from injured myocardial tissues, and mitochondrial functions (mitochondrial superoxide production, membrane potential and ATP production) determined. The neonatal rat cardiomyocytes were isolated and infected with KLK1 recombinant adenovirus to achieve KLK1 overexpression, and then hypoxia/reoxygenation (H/R) cell model was established. The H/R cells were treated with the media containing bradykinin receptor type 1 (B1R) antagonist R715 or bradykinin receptor type 2 (B2R) antagonist HOE140. The cell viability was determined with MTT assay, and the mitochondrial functions were observed. Results In I/R rats, KLK1 overexpression could alleviate the cardiac injury, and decrease infarction area and cell apoptosis (all P<0.01) in cardiac ischemic risk area; moreover, KLK1 overexpression could improve mitochondrial dysfunction, decrease mitochondrial peroxide production, and increase mitochondrial membrane potential and ATP production (all P<0.01). In vitro cardiomyocyte model, KLK1 overexpression could also attenuate cardiomyocyte injury (P<0.01) and mitochondrial dysfunction (P<0.05, P<0.01), which could be inhibited by B2R antagonist HOE140. Conclusion KLK1 mitigates mitochondrial dysfunction after cardiac I/R injury, which may be an important mechanism of its cardioprotective effect.