Diabetic cardiomyopathy (DCM) is a complication of diabetes, which may be associated with a great risk of mortality. Comprehensively understanding the effects and mechanisms of acetylshikonin (AS) on the attenuation of DCM will be helpful for the innovative drug investigation and clinical application. This study aimed to explore the possible links and signaling pathways between AS and DCM. A cohort of diabetic mice was established based on disease model (Model, AS, Control groups), and their serum samples were collected for non-targeted metabolomics. The high resolution mass spectrometry data among the three groups were analyzed by combining multivariate and univariate statistical methods. The differential metabolites and altered metabolic pathways were screened out and identified by examining the Kyoto Encyclopedia of Genes and Genomes database, respectively.