Abstract: At present, effective policy measures to improve agricultural eco-efficiency is a major practical issue widely addressed by governments and academia. Based on panel data from 238 prefecture-level administrative regions in China from 2002 to 2022, in this study, we used the super-efficiency slacks-based measure (SBM) model to measure agricultural eco-efficiency and reveal its spatial and temporal evolution characteristics. Using the multi-time-point difference-in-differences model, the pilot zone of agricultural green development was considered a quasi-natural experiment to explore its impact, heterogeneity, and regulatory mechanisms on agricultural eco-efficiency. The results showed the following: 1) China’s agricultural eco-efficiency increased from 0.039 in 2002 to 0.175 in 2022, showing a dynamic trend of annual increase. The clustering area of high agricultural eco-efficiency was dominated by South China and evolved into a spatial pattern of multi-point flowering in Sichuan and Chongqing, Central China, Western China, Northeast China, and South China. 2) Benchmark regression results showed that compared with non-pilot zones and zones before pilot implementation, pilot zones had a significant positive impact on the improvement of agricultural eco-efficiency, and the results had good robustness. 3) Regional differentiation analysis revealed that the impact of pilot zones on agricultural eco-efficiency was more significant in the eastern, coastal, and major grain-producing areas. 4) The results of the adjustment mechanism test showed that agricultural industry agglomeration and diversification of the crop planting structure played a positive role in improving agricultural eco-efficiency in the pilot zone. Finally, we propose countermeasures and suggestions for the orderly promotion of pilot zones and the improvement of agricultural eco-efficiency, including establishing a technology sharing platform for enhancing agricultural eco-efficiency, building a differentiated regional promotion system, and creating a policy reinforcement mechanism driven by the “dual engines” of industrial agglomeration and crop diversification.