BackgroundThe research on the gut microbiota of neonates and preterm neonates by conventional feeding methods is still limited.ObjectiveThis study aimed to systematically characterize gut microbiota variations across three feeding methods in neonates, with a specific focus on preterm neonates.MethodsThis study included 265 neonates from the Hunan Children’s Hospital cohort, categorized by feeding methods into breast-fed (BF, n = 122), formula-fed (FF, n = 86), and mixed-fed (MF, n = 57) groups. Additionally, the analysis was further stratified into preterm and full-term neonates to investigate the relationship between feeding methods and gut microbiota. Gut microbiota composition was assessed using 16S rRNA gene sequencing of neonatal fecal samples. Microbial patterns associated with feeding methods were identified through MaAsLin2 analyses.ResultsUnadjusted analyses showed that compared with the FF and MF groups, the BF group exhibited significantly higher relative abundances of Bifidobacterium (p = 0.018) and Staphylococcus (p = 0.039). In addition, the relative abundance of Akkermansia (p = 0.046) in the FF group was higher than that in the other two groups. MaAsLin2 analyses revealed the FF group had higher relative abundances of Verrucomicrobiota (Coef = 1.46, p = 0.001, FDR q-value = 0.055) and Akkermansia (Coef = 1.53, p = 0.001, FDR q-value = 0.040) than those in the BF group among all neonates, particularly preterm neonates. The BF neonates displayed enriched Staphylococcus (Coef = −1.47, p = 0.009, FDR q-value = 0.154) and depleted Rothia (Coef = 1.45, p = 0.004, FDR q-value = 0.121). The MF group showed higher Streptococcus abundance relative to the BF group (Coef = 0.95, p = 0.019, FDR q-value = 0.204). Functional prediction indicated potential group differences in propionate metabolism and degradation of valine, leucine, and isoleucine pathways, particularly in preterm neonates.ConclusionOur study demonstrates an association between feeding methods and gut microbiota composition in neonates. The BF neonates exhibited increased relative abundance of Staphylococcus and decreased relative abundances of Akkermansia and Rothia compared with FF neonates. The MF group had higher relative abundance of Streptococcus than BF group. Furthermore, among preterm neonates, FF group was associated with a higher abundance of Akkermansia compared with BF group.