Autophagy has been proved to play a vital role in cardiac hypertrophy. The present study was designed to investigate the relationship between miR-100-5p and autophagy in the development of cardiac hypertrophy. Here, miR-100-5p expression was detected in abdominal aortic coarctation (AAC)-induced cardiac hypertrophy rats and Angiotensin II (Ang II)-stimulated cardiomyocytes. In vitro and in vivo experiments were performed to explore the function of miR-100-5p on autophagy and cardiac hypertrophy. We also investigated the mechanism of miR-100-5p on autophagy with dual-luciferase reporter assays, RNA immunoprecipitation (RIP), quantitative real-time PCR (qRT-PCR), western blot, immunofluorescence, and transmission electron microscopy (TEM). The results showed that miR-100-5p was highly expressed in hypertrophic hearts and Ang II-induced cardiomyocytes. Overexpression of miR-100-5p promoted the expression of cardiac hypertrophy markers ANP, BNP and β-MHC and cell surface area, while those were suppressed by miR-100-5p inhibitor. Knockdown of miR-100-5p by antagomiR significantly improves cardiac function and attenuate cardiac hypertrophy in vivo. Mechanistic investigation has found that miR-100-5p promote autophagy by targeting mTOR. Inhibition of autophagy by 3-methyladenine (3-MA) or mTOR overexpression could reverse the function of miR-100-5p in cardiac hypertrophy. These results elucidate that miR-100-5p promoted the pathogenesis of cardiac hypertrophy through autophagy activation by targeting mTOR.