Abstract: Objective To investigate the role of intestinal fungi in the progression of heart failure (HF) associated with chronic kidney disease (CKD). Methods This study consisted of two parts. The first part was a clinical study. Fecal samples from CKD patients (CKD group), CKD patients with HF (CKD+HF group), and healthy individuals (healthy control group) were subjected to 18S rRNA sequencing to compare differences in intestinal fungal microbiota among the three groups. The second part was an animal experiment. Male C57BL/6J mice were randomly divided into a control group (fed a standard diet), a CKD group (fed a 0.2% adenine diet), and a CKD+amphotericin B group (fed a 0.2% adenine diet + 0.5 mg/L amphotericin B in drinking water), with 10 mice in each group. After successful modeling, cardiac function and histomorphological differences among the three groups were compared by assessing exercise tolerance, left ventricular ejection fraction (LVEF), left ventricular shortening fraction (LVFS), serum N-terminal pro-brain natriuretic peptide (NT-proBNP) levels, and histological examinations (HE staining and Masson staining) of cardiac tissue. Results The clinical study revealed that compared with the healthy control group (n=18) and the CKD group (n=17), the relative abundance of Ascomycota phylum in the intestines of the CKD+HF group (n=18) was significantly increased (P=0.007, P=0.009), along with significant increases in the relative abundances of Desmodorida and Candida-Lodderomyces clade (P=0.005, P=0.003). In the animal experiment, compared with the control group, the CKD group exhibited significantly reduced exercise tolerance, LVEF, and LVFS (all P<0.001), elevated serum NT-proBNP levels (P<0.001), and disordered cardiomyocyte arrangement with increased fibrosis (P<0.001) as shown by HE and Masson staining. In contrast, compared with the CKD group, the CKD+amphotericin B group demonstrated significant improvements in exercise tolerance, LVEF, and LVFS (all P<0.001), reduced NT-proBNP levels (P<0.001), and well-arranged cardiomyocytes with markedly decreased myocardial fibrosis (P<0.001). Conclusions Intestinal fungi may be associated with the progression of HF in CKD patients. Depletion of intestinal fungi could potentially ameliorate cardiac remodeling and delay the onset and progression of HF. Intestinal fungi may serve as a novel therapeutic target for HF in CKD patients.