Rationale: Diabetes mellitus is often associated with cardiovascular complications, which is the leading cause of morbidity and mortality among patients with diabetes mellitus, but little is known about the mechanism that connects diabetes mellitus to the development of cardiovascular dysfunction. Objective: We aim to elucidate the mechanism underlying hyperglycemia-induced cardiac dysfunction on a well-established db/db mouse model for diabetes mellitus and diabetic complications that lead to heart failure. Methods and Results: We first profiled the expression of microRNAs (miRNAs) by microarray and quantitative reverse transcription polymerase chain reaction on db/db mice and identified miR-320 as a key miRNA associated with the disease phenotype. We next established the clinical relevance of this finding by showing the upregulation of the same miRNA in the failing heart of patients with diabetes mellitus. We demonstrated the causal role of miR-320 in inducing diabetic cardiomyopathy, showing that miR-320 overexpression exacerbated while its inhibition improved the cardiac phenotype in db/db mice. Unexpectedly, we found that miR-320 acts as a small activating RNA in the nucleus at the level of transcription. By chromatin immunoprecipitation sequencing and chromatin immunoprecipitation quantitive polymerase chain reaction analysis of Ago2 (argonaute RISC catalytic component 2) and RNA polymerase II in response to miR-320 induction, we identified CD36 (fatty acid translocase) as a key target gene for this miRNA and showed that the induced expression of CD36 is responsible for increased fatty acid uptake, thereby causing lipotoxicity in the heart. Conclusions: These findings uncover a novel mechanism for diabetes mellitus-triggered cardiac dysfunction, provide an endogenous case for small activating RNA that has been demonstrated to date only with synthetic RNAs in transfected cells, and suggest a potential strategy to develop a miRNA-based therapy to treat diabetes mellitus-associated cardiovascular complications.