China’s agricultural green development is of great significance to protecting the ecological environment, ensuring food security, and promoting rural revitalization. This study considers China’s agricultural green development pilot zones policy as a natural experiment to examine its impact on regional agricultural eco-efficiency. Based on county-level data in Hubei Province from 2013 to 2021, the EBM-Undesirable model was employed to measure regional agricultural eco-efficiency. Then, the staggered DID model was conducted to estimate the policy impact. The results indicate that the pilot zones policy significantly enhances the agricultural eco-efficiency in the designated areas. The results from heterogeneity and mechanism analysis indicate that the pilot zones policy has a more substantial effect on the western region and less-affluent counties, and rural innovation serves as an intermediary variable for the policy to exert the “green” effect. Therefore, it is crucial for the government to consider regional disparities and the role of rural innovation when advancing policy implementation. Tailoring measures to suit specific local circumstances and consistently enhancing agricultural eco-efficiency in different regions is essential.