Background <p>Coronary Microvascular Dysfunction (CMD) represents a critical pathological substrate for ischemic heart disease and is strongly associated with major adverse cardiovascular events. Zacopride, known for its dual cardiovascular regulatory properties targeting the 5-HT₄ receptor and Kir2.1 channel, lacks evidence regarding its systemic impact on the gut microbiota-metabolism axis. Therefore, this study aims to elucidate the structural and metabolic characteristics of gut bacteria and fungi in CMD, and to explore the multidimensional therapeutic mechanisms of Zacopride through "microbial remodeling-metabolic regulation-microcirculation repair."</p> Methods <p>Sixty Sprague–Dawley rats were randomized into three groups: coronary microvascular dysfunction (CMD), healthy control (NC), and Zacopride intervention (ZAC). CMD and ZAC groups received high-fat diet plus streptozotocin (STZ, 35&#xa0;mg/kg) for modeling. ZAC rats were orally administered 5&#xa0;mg/kg Zacopride daily for 7&#xa0;days. Transthoracic Doppler echocardiography measured left anterior descending coronary artery resting/stress peak flow velocity and coronary flow reserve (CFR). Ileocecal contents underwent bacterial-fungal metagenomic sequencing to identify differential metabolic pathways. Spearman's correlation assessed cross-kingdom ecological interactions. Nine machine learning algorithms constructed classification models, with Random Forest (RF) and an optimal model identifying key genera. Linear Discriminant Analysis Effect Size validated microbial biomarkers.</p> Results <p>Zacopride partially restored the CFR in CMD rats, demonstrating a therapeutic effect, and exerted a beneficial influence on the structure and diversity of the gut microbiota. The CMD state significantly reduced the expression levels of the Chlorophyll a and tryptophan metabolic pathways in the gut microbiota. Zacopride specifically restored the Chlorophyll a pathway but did not significantly recover the tryptophan metabolic pathway. RF and Elastic Net (ENET) identified <i>JC017</i>, <i>Chromelosporium</i>, and <i>Barnesiella</i> as biomarker microbiota for CMD. Notably, <i>JC017</i> primarily mediate the therapeutic effects of Zacopride via direct or indirect modulation of the Chlorophyll a metabolic pathway. <i>Chromelosporium</i>, acting as an interactive hub between fungi and bacteria, formed a cross-kingdom symbiotic relationship with <i>Bradyrhizobium</i>. Additionally, the reduction in <i>Barnesiella</i> abundance constitutes a distinctive feature of gut microbial dysbiosis in CMD.</p> Conclusion <p>This study provides the first evidence that the gut microbiota modulates the pathogenesis of CMD through the "chlorophyll/heme-tryptophan metabolic axis." Furthermore, we demonstrate that Zacopride exerts therapeutic effects by remodeling microbiota-host interactions and regulating this metabolic axis, revealing a novel mechanistic link between microbial metabolism and CMD progression.</p> Graphical abstract <p></p>