Diabetic cardiomyopathy (DCM) is a chronic metabolic disease with few effective therapeutic options. Immunoproteasome is an inducible proteasome that plays an important role in the regulation of many cardiovascular diseases, while its role in DCM remains under discussion. The present study aims to demonstrate whether inhibiting immunoproteasome subunit low molecular weight polypeptide 7 (LMP7) could alleviate DCM. Here, we established a type I diabetes mellitus mouse model by streptozotocin (STZ) in 8-week-old male wild-type C57BL/6J mice. We found that immunoproteasome subunit LMP7 was overexpressed in the heart of diabetic mice, while inhibiting LMP7 with pharmacological inhibitor ONX0914 significantly alleviated myocardial fibrosis and improved cardiac function. Besides, compared with diabetic mice, ONX0914 treatment reduced protein levels of mesenchymal markers (Vimentin, α-smooth muscle actin, and SM22α) and increased endothelial markers (VE-cadherin and CD31). In TGFβ1 stimulated HUVECs, we also observed that ONX0914 could inhibit endothelial-mesenchymal transition (EndMT). Mechanistically, we prove that ONX0914 could regulate autophagy activity both in vivo and vitro. Meanwhile, the protective effect of ONX0914 on TGFβ1 stimulated HUVECs could be abolished by 3-methyladenine (3MA) or hydroxychloroquine (CQ). All in all, our data highlight that inhibition of LMP7 with ONX0914 could ameliorate EndMT in diabetic mouse hearts at least in part via autophagy activation. Thus, LMP7 may be a potential therapeutic target for the DCM.© 2023 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.