Establishing the gut microbiome during early life is the first critical interaction between perinatal influences and the physiological adaptations of the newborn. This study examined the effects of mode of delivery on the gut microbiome composition and functional potential and the temporal maturation of the gut microbiome within the first weeks after birth. Analyzing early stool specimens of neonates born vaginally and via cesarean sections showed that mode of delivery creates a lasting imprint on the gut microbiome, producing distinct community structures characterized by varying quantities of specific pioneering microbes. The functional pathways prediction showed differing pathways enrichment based on mode of delivery, with vaginal deliveries favoring carbohydrate metabolism and production of short-chain fatty acids, and cesarean deliveries associated with stress response and aerobic metabolism. Further, longitudinal studies showed diverged trajectories in gut microbiome maturation, indicating that cesarean delivery is associated with more variability and a longer microbial developmental lag. The merger of compositional, functional, and temporal information, supports a systems-level hypothesis in which the mode of delivery influences early microbial ecosystems and the physiological adaptations of the newborn. The study exemplifies the importance of early microbial colonization in defining the subsequent health of the neonate and illustrates the likely importance of microbiome-informed strategies in perinatal care.