<p>This study aimed to explore whether Fufangteng Yixin Formula (FFTYXF) can ameliorate myocardial ischemia-reperfusion injury (MIRI) by regulating the gut microbiota and resultant metabolites. Wistar rats were given FFTYXF by gavage for 7 days, and MIRI rat model was established. Serum level of inflammatory markers was determined by ELISA, and heart function was evaluated by echocardiography. Myocardial histological changes and infarct size were examined by hematoxylin-eosin and triphenyltetrazolium chloride staining, respectively. 16S rRNA gene and metagenomics analyses were employed to explore gut microbiota, while untargeted metabolomics analysis was used to explore serum metabolites. FFTYXF pretreatment could significantly improve cardiac function, reduce infarct size, decrease level of inflammatory factors (TNF-α and IL-6) and inflammatory cells infiltration. At genus level, g__<i>Oscillibacter</i> and g__<i>Rikenellaceae</i>_<i>RC9</i>_<i>gut</i>_<i>group</i> were identified as key microbial bacteria in MIRI rat response to FFTYXF pretreatment. After FFTYXF pretreatment, the functional categories of gut microbiota were participated in fatty acid (FA) biosynthesis/metabolism, glycolysis _ gluconeogenesis and sphingolipid metabolism. Genes response to FFTYXF pretreatment in MIRI rats included K00023 (phbB), K00281 (GLDC, gcvP), K03879 (ND2), K07827 (KRAS) and K15192 (BTAF1), and they were mainly participated in carbon, butanoate, glyoxylate and dicarboxylate metabolism. Differential metabolites were also mainly participated in FA biosynthesis/metabolism, such as alpha-Linolenic acid, omega-3/omega-6 FA, and flavan-3-ol metabolic pathway. Abundance of g__<i>Rikenellaceae</i>_<i>RC9</i>_<i>gut</i>_<i>group</i> positively correlated with differential metabolites FAHFA 34:0, FAHFA 16:1/18:3, and FA 24:5. FFTYXF could alleviate MIRI by modulating gut microbial bacteria alteration and resultant metabolites, particularly short-chain FAs.</p> Graphical abstract <p></p>