IntroductionThe microbiota-gut-brain axis is implicated in Alzheimer’s disease. Gut microbiota alterations in mild cognitive impairment (MCI) are inconsistent and remain to be understood. This study aims to investigate the gut microbial composition associated with MCI, cognitive functions, and structural brain differences.MethodsA nested case-control study was conducted in a community-based prospective cohort where detailed cognitive functions and structural brain images were collected. Thirty-one individuals with MCI were matched to sixty-five cognitively normal controls by age strata, gender, and urban/rural area. Fecal samples were examined using 16S ribosomal RNA (rRNA) V3–V4 sequencing. Compositional differences between the two groups were identified and correlated with the cognitive functions and volumes/thickness of brain structures.ResultsThere was no significant difference in alpha and beta diversity between MCIs and cognitively normal older adults. However, the abundance of the genusRuminococcus,Butyricimonas, andOxalobacterdecreased in MCI patients, while an increased abundance of nine other genera, such asFlavonifractor, were found in MCIs. Altered genera discriminated MCI patients well from controls (AUC = 84.0%) and were associated with attention and executive function.ConclusionThis study provides insights into the role of gut microbiota in the neurodegenerative process.