Rationale Although a growing body of evidence indicates that the scores of cognitive function in hemodialysis patients are significantly lower than those of healthy individuals, underlying mechanisms have not been fully elucidated. Objectives To investigate the roles of gut microbiota and serum metabolites in hemodialysis patients with mild cognitive decline (MCD). Methods A total of 30 healthy individuals and 77 hemodialysis patients were enrolled and were classified into healthy control (HC), normal cognitive function (NCF), and MCD groups by evaluation of Montreal Cognitive Assessment. Fecal samples were analyzed by 16S rRNA and serum samples were analyzed by gas chromatography-mass spectrometry from all subjects. Results The 16S rRNA study demonstrated that the gut microbiota profiles, including alpha- and beta-diversity, and a number of 16 gut bacteria were significantly altered in the MCD group compared with those in HC or those with NCF. Astudy showed that a total of 29 serum metabolites were altered in the MCD group. Receiver operating characteristic curves showed that GenusBilophilaand serum putrescine might be sensitive biomarkers to indicate MCD in patients with hemodialysis. Conclusions These findings demonstrate gut microbiota and serum metabolites were probably involved in the pathogenesis of hemodialysis-related MCD. Therapeutic strategies targeting abnormalities in gut microbiota and serum metabolites may facilitate the beneficial effects for hemodialysis patients with MCD.