To investigate the effect and mechanism of microRNA‐186‐5p (miR‐186‐5p) on the apoptosis in high glucose (HG)–treated cardiomyocytes. Diabetic cardiomyopathy model was established in cardiomyocytes by stimulating with HG. The expressions of miR‐186‐5p and toll‐like receptor 3 (TLR3) were detected by quantitative polymerase chain reaction or Western blot analysis, respectively. Apoptosis was detected in HG‐treated cardiomyocytes by flow cytometry and Western blot analysis. The interaction between miR‐186‐5p and TLR3 was explored by bioinformatics analysis and luciferase activity assay. Results showed that miR‐186‐5p expression was downregulated in HG‐treated cardiomyocytes and its overexpression reversed HG‐induced apoptosis and cleaved caspase‐3 protein expression. Moreover, TLR3 was indicated as a target of miR‐186‐5p and regulated by miR‐186‐5p. Knockdown of TLR3 suppressed HG‐induced apoptosis and cleaved caspase‐3 protein expression. Besides, restoration of TLR3 ablated the effect of miR‐186‐5p on cell apoptosis. Collectively, miR‐186‐5p attenuated HG‐induced apoptosis by regulating TLR3 in cardiomyocytes, providing novel biomarker for treatment of diabetic cardiomyopathy.