Objective To investigate the expression levels of microRNA-186-5p (miR-186-5p) and Toll-like receptor 3 (TLR3) and their relationships with the apoptosis in high-glucose (HG)-treated AC16 cardiomyocytes. Methods Target Scan7.1 database predicted that miR-186-5p could act directly on TLR3. Diabetic cardiomyopathy model was established in cardiomyocytes stimulated by HG. The expression of miR-186-5p was detected by real-time quantitative PCR and the expression of TLR3 was detected by Western blot analysis. The expression of miR-186-5p or TLR3 was enhanced or reduced by cell transfection. The apoptosis of cardiomyocytes was detected by flow cytometry. The expression of cleaved caspase-3(c-caspase-3) was detected by Western blot analysis, and the interaction between miR-186-5p and TLR3 was analyzed by luciferase activity assay. Results The bioinformatics analysis and luciferase activity assay showed that TLR3 was a direct target gene of miR-186-5p. The expression of miR-186-5p was down-regulated in HG-treated cardiomyocytes, and the over-expression of miR-186-5p reversed HG-induced cardiomyocyte apoptosis and reduced the protein level of c-caspase-3. Down-regulation of TLR3 inhibited HG-induced apoptosis and reduced protein level of c-caspase-3 in cardiomyocytes. Over-expression of TLR3 increased HG-induced cardiomyocyte apoptosis and reversed the effect of miR-186-5p. Conclusion The miR-186-5p can inhibit the apoptosis of cardiomyocytes induced by HG via down-regulating TLR3 expression.