Analyzing the carbon stock in a terrestrial ecosystem is a key link for research on the global and regional carbon cycle. Assessing the long-time-series carbon stock in the West Dongting Lake National Nature Reserve will provide scientific data for regional ecological monitoring and management. Based on the land use data from 2000 to 2020, this study explored the spatiotemporal changes in the carbon stock of the nature reserve based on the carbon stock estimated using the InVEST model and identified key areas of carbon stock changes. The results indicate that in the past two decades, the carbon stock in the nature reserve exhibited a fluctuating upward trend, ranging from 113.5×10⁴ to 125.7×10⁴ tons. The carbon stock presented relative changing rates of less than 2% during this period, except for 2003, when the changing rate was 3.2%. Over the past two decades, the core zone of the nature reserve ranked first in carbon stock among subregions every year, followed by the pilot zones. The carbon stock in most areas of the nature reserve remained unchanged or changed slightly. Nevertheless, there still existed some areas with significant changes in the carbon stock. The key areas of carbon stock changes featured diverse spatial distribution patterns of carbon stock, such as concentrated, linear, and scattered patterns, with land use types in these areas exhibiting corresponding change intensities of carbon stock. The changes in the carbon stock in the pilot zones were greatly affected by human interference, while those in the core area were primarily related to precipitation. The results of this study will assist in scientifically promoting carbon neutrality and peak carbon dioxide emissions in the West Dongting Lake National Nature Reserve.