This study examines the Holocene stratigraphic record and paleoenvironmental evolution of a large estuarine system, at high temporal and spatial resolution, in the context of changing climate, sea level, and hydrodynamics. New data are used to examine two time periods of increased marine influence within Pamlico Sound in northeastern North Carolina interpreted to be the result of extensive barrier island segmentation synchronous with periods of rapid climate change during the late Holocene. The study reveals the controls on the stratigraphic development and the highly dynamic character of the coastal system in response to climate, and sea-level change as it interacts with paleotopography. These findings can be used to inform projections of future environmental changes. Estuarine waters occupied paleovalleys in the Pamlico Sound region as early as ca. 7500 cal BP and Pleistocene interfluves separated the paleovalleys from the Atlantic Ocean until ca. 5500 cal BP, when they were flooded by rising sea-level forming the broad, shallow Pamlico Sound. Initial barrier islands and shoals likely formed sometime between 5500 and 5000 cal BP, providing the geomorphic setting for continued estuarine conditions behind the barriers. At ca. 4000 cal BP, an increase in marine influence and sand content is detected in multiple cores, and is correlated to seismic data within the northern Pamlico Sound basin. This change is interpreted to be the result of increased segmentation of barrier islands (thus a greater number of inlets, or wider inlets), which is consistent with a rapid transgression seen in other areas of the U.S. east coast. The segmentation may have been partially facilitated by a rapid increase in lagoonal area and tidal prism by overtopping of interfluves. Greater barrier island continuity is evident from 3500 to 1200 cal BP, as indicated by muddy deposits with low brackish estuarine foraminiferal assemblages. At ca. 1200 cal BP, seismic, sedimentological and micropaleontological data suggest an increase in wave and current energy and marine influence throughout southeastern 3 Pamlico Sound. These changes are interpreted to represent extensive segmentation of the barrier islands during the Medieval Climate Anomaly (MCA). Since ca. cal BP, the Pamlico Sound system has returned to a more restricted state, as inlets have closed. Currently only three major inlets segment the Outer Banks, the barrier island system fronting Pamlico Sound, but warming climate and increasing rates of sea-level rise suggest another episode of barrier island segmentation began about a half century ago and appears that it may extend into the near future.