The purpose of this study was to elucidate the regulatory role of microRNA-324-5p (miRNA-324-5p) in inhibiting inflammatory response of diabetic vessels by regulating CPT1A level, thus alleviating the development of type 2 diabetes mellitus (T2DM). Arterial vessels (splenic artery) and serum exosomes were extracted from 30 T2DM patients and 30 non-T2DM subjects treated in Binzhou People's Hospital from 2015 to 2019. Relative levels of miRNA-324-5p and CPT1A in each subject were detected. Then, VSMCs were induced with high-glucose, followed by detection of inflammatory factor levels. Next, the regulatory effects of miRNA-324-5p and CPT1A on viability, 5-Ethynyl-2'-deoxyuridine (EdU)-positive ratio, and release of inflammatory factors in VSMCs were determined. Finally, Dual-Luciferase reporter assay was conducted to verify the interaction between miRNA-324-5p and CPT1A. The results revealed that compared with non-T2DM subjects, miRNA-324-5p was downregulated in splenic arteries and exosomes in T2DM patients. High-glucose treatment in VSMCs triggered the release of the inflammatory factors. In addition, the overexpression of miRNA-324-5p in VSMCs reduced viability and inflammatory factor levels, and the inhibited trends were partially reversed by overexpression of CPT1A. CPT1A was indicated to be the target gene binding miRNA-324-5p. MiRNA-324-5p exerts an inhibitory effect on T2DM-induced inflammation in blood vessels by negatively regulating CPT1A level and reducing the release of inflammatory factors. MiRNA-324-5p might be a promising therapeutic target for T2DM.