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Sepsis is a life-threatening condition in preterm infants. Neonatal microbiota plays a pivotal role in the immune system maturation. Changes in gut microbiota have been associated to inflammatory disorders; however, a link with sepsis in the neonatal period has not yet been established. We aimed to analyze gut microbiota and mucosal gene expression using non-invasively obtained samples to provide with an integrative perspective of host-microbe interactions in neonatal sepsis. For this purpose, a prospective observational case-control study was conducted in septic preterm dizygotic twins and their non-septic twin controls. Fecal samples were used for both microbiota analysis and host genome-wide expression using exfoliated intestinal cells. Gene expression of exfoliated intestinal cells in septic preterm showed an induction of inflammatory and oxidative stress pathways in the gut and pro-oxidant profile that caused dysbiosis in the gut microbiota with predominance of Enterobacteria and reduction of Bacteroides and Bifidobacterium spp.in fecal samples, leading to a global reduction of beneficial anaerobic bacteria. Sepsis in preterm infants induced low-grade inflammation and oxidative stress in the gut mucosa, and also changes in the gut microbiota. This study highlights the role of inflammation and oxidative stress in neonatal sepsis on gut microbial profiles. ; M.C. acknowledges a Research Grant Fellowship "Rio Hortega" and M.V. a RD12/0022/0012 (Red SAMID) and FIS PI14/0443 grants from the Instituto Carlos III (Spanish Ministry of Economy and Competitiveness) and Fondos FEDER from the European Union. C.B., M.C.C. and G.P.M. acknowledge the grant AGL2013-47420-R and by the Fun-C-Food CSD2007-00063 project from the Consolider-Ingenio program, both from the Spanish Ministry of Science and Innovation. ; Peer reviewed