Objective: Development of the gastrointestinal tract and immune system can be modulated by the gut microbiota. Establishment of the intestinal microbiota, in its turn, is affected by host and environmental factors. As such, development of the gut microbiota is greatly impacted in preterm infants, who have an immature gut and are exposed to factors like hospitalization, caesarean section, antibiotics, and respiratory support. Design: We analyzed fecal microbiota composition and activity of ten preterm infants (gestational age 25-30 weeks; birthweight 630-1750 g) during the first six postnatal weeks through metaproteomics (LC-MS/MS) and 16S-rRNA gene sequencing. Results: A gestational-age-dependent microbial signature is observed, enabling microbiota-based differentiation between extremely preterm (25-27 weeks gestation) and very preterm (30 weeks gestation) infants. In very preterm infants, the intestinal microbiota developed toward a Bifidobacterium-dominated community and was associated with high abundance of proteins involved in carbohydrate and energy metabolism. Extremely preterm infants remained predominantly colonized by facultative anaerobes and were associated with proteins involved in membrane transport and translation. Delayed colonization by obligate anaerobes could be associated with antibiotic treatment and respiratory support. Conclusion: We speculate that gestational age and its associated intensity of care (e.g. antibiotics and respiratory support) affects intestinal microbiota composition and activity in preterm infants. As the gut microbiota plays a major role in development of the neonate, gestational age and its associated factors could set the stage for early and later life health complications via interference with microbiota development.