Changes in the value of natural ecosystem regulating services over a period of time can reflect the effectiveness of regional ecological protection measures. Qiandongnan Miao-Dong Autonomous Prefecture of Guizhou Province is located in the karst region of southwest China, which with its high forest coverage is an important ecological barrier in the upper reaches of the Yangtze River and the Pearl River. Qiandongnan Prefecture was chosen as a case study; we constructed an evaluation index system based on gross ecosystem product (GEP) and applied the methods of value accounting to ecosystem regulating services to discuss the application of GEP-based accounting in assessing the effectiveness of ecological protection measures. The evaluation results showed that the ecosystem regulating services value of Qiandongnan in 2010 was 223.878 billion Yuan, which was 7.16 times its GDP. Ecosystem regulating service value per unit area and per capita in the national key ecological function area counties are higher than those in non-key ecological function area counties. From 2000 to 2010, the value of ecosystem regulating services in Qiandongnan Prefecture increased by 2.26% at constant price; the rate of increase of this value in non-key ecological function area counties was higher than that in key ecological function area counties. Our results suggest that the value of ecosystem regulating services can directly reflect the status and changes in the ecological environment during a certain period in Qiandongnan Prefecture, and highlight the effect of comprehensive implementation of projects for ecological protection. The value of ecosystem regulating services provides a scientific basis for subsequent implementation of ecological protection measures in Qiandongnan Prefecture.