Abstract: Myocardial fibrosis is a crucial pathological process of multiple cardiovascular diseases, yet effective therapies remain limited. Levistilide A (LA) is a bioactive constituent derived from the dried rhizome of Ligusticum chuanxiong Hort. (known as Chuanxiong in traditional Chinese medicine) with potential cardioprotective activities, but its therapeutic efficacy against myocardial fibrosis and the underlying molecular targets remain elusive. Here, we show that LA inhibits TGF-β1-induced fibroblast transformation and proliferation in vitro and alleviates myocardial fibrosis in a mouse model following myocardial infarction. By using drug affinity responsive target stability (DARTS), proteomics, and surface plasmon resonance (SPR) approaches, we identify mitochondrial malate dehydrogenase 2 (MDH2) as a putative molecular target of LA. Mechanistically, LA directly binds to MDH2 and suppresses its TGF-β1-induced phosphorylation, thereby inhibiting MDH2 enzymatic activation. This leads to the blockage of the malate-aspartate shuttle (MAS), thereby suppressing the enhanced mitochondrial oxidative phosphorylation (OXPHOS) and ultimately inhibiting fibroblast transformation and myocardial fibrosis. Therefore, our findings reveal that LA alleviates myocardial fibrosis through targeting MDH2-mediated mitochondrial metabolic reprogramming, highlighting MDH2 as a potential therapeutic target and LA as an active compound in the treatment of myocardial fibrosis.