Diabetic cardiomyopathy is characterized by the deterioration of the myocardial function. Emerging evidences have indicated that leukocytic toll-like receptor 2 (TLR2) played an important role in the development of diabetic cardiomyopathy. Our study aimed to investigate whether TLR2 knockout (KO) exerted a cardioprotective effect in vivo. The establishment of diabetes model was set up in mice via intraperitoneal injection of streptozotocin (STZ). Results demonstrated that blocking of TLR2 significantly suppressed the enhanced left ventricular end-diastolic dimension (LVEDD), left ventricular end systolic diameter (LVESD) and the reduced the heart rate in diabetic cardiomyopathy mice. The decreased resting cell length, PS, TPS and + dL/dt while increased TR90 and - dL/dt caused by diabetic cardiomyopathy were remarkably inhibited by TLR2 KO. Besides that, the alleviated ΔFFI (360/380), decreased SERCA2a and p-NFATc3 expressions, extended Ca2+ decay time and elevated Calcineurin A induced by diabetic cardiomyopathy were vastly repressed by TLR2 KO in cardiocytes. Moreover, TLR2 gene silence could ameliorate oxidative stress-induced apoptosis, evidences were the up-regulated superoxide generation and Bax/Bcl-2 expression while restrained GSH/GSSG ratio caused by diabetic cardiomyopathy were tremendously repressed in TLR2 KO mice. Furthermore, blocking of TLR2 remarkably attenuated the augmented fibrosis areas of heart tissues in mice with diabetic cardiomyopathy. The result of the enhanced α-SMA and collagenⅠ caused by diabetic cardiomyopathy were suppressed in heart tissues of TLR2 KO mice further validate it. All in all, our study demonstrated that diabetes-induced cardiac dysfunction could be attenuated by TLR2 KO.