Long-term trends and relationships between large mesozooplankton (1.5–20 mm) and the physical environment were used to investigate impacts of climate change and variability in the North Inlet estuary, South Carolina. Biweekly collections (365 μm) from 1981 to 2003 demonstrated distinct seasonal patterns and large interannual fluctuations in abundances. Significant long-term decreases in abundances were observed for total zooplankton and 12 taxa (e.g., Gobiosoma larvae, chaetognaths, and adult shrimps), whereas 14 others, including some of the most abundant constituents, did not change (e.g., mysids, amphipods, crab megalopae, and Palaemonetes larvae). The composition of the fauna was similar at the beginning and end of the 23-year period. A significant long-term increase in winter water temperature (2.6°C) and a decrease in summer salinity (3.2) were determined. Relationships between physical conditions and taxa varied in strength and direction, and most did not agree with their long-term trends. However, some major taxa appeared to respond to climate variability, including the timing and intensity of El Niño or La Niñ a events. Although changes in the North Inlet mesozooplankton were minor compared to fauna in northern waters in recent decades, a trend toward the freshening and warming of this system could alter processes including larval recruitment, secondary production, and trophic interactions. The design of long-term sampling programs must consider effects of tide and time of day on zooplankton. The National Estuarine Research Reserve System provides an ideal platform for the collection of time series measurements that are critical to the understanding of climate change on biological communities.