Abstract: With increasingly severe agricultural pollution, it is important to explore the effects of agricultural science and technology investments on agricultural ecological efficiency to alleviate rural ecological pressure and promote the healthy development of rural areas. This study used a super-efficiency slack-based measure (SBM) model to measure the agricultural ecological efficiency of provinces in eastern, middle, and western China from 2000 to 2018. According to the Moran index, the spatial autocorrelation of agricultural ecological efficiency and agricultural science and technology input were analyzed. The spatial spillover effect and threshold characteristics of the impact of agricultural science and technology input on agricultural ecological efficiency were explored by using the spatial econometric model. Results showed that from 2000 to 2018, the agro-ecological efficiency was high in the eastern and western regions and low in the middle region. The agro-ecological efficiency in the eastern, middle, and western regions fluctuated significantly from 2000 to 2018, with slight fluctuations in 2000–2003. Agricultural ecological efficiency declined slightly from 2004 to 2008 and then rose slightly from 2008 to 2010. In 2010, the agricultural ecological efficiency was 0.731, after which it declined slightly from 2011 to 2014. From 2015 to 2017, the national agricultural ecological efficiency dropped to 0.5894, 0.5839, and 0.5159, respectively. In 2018, the annual agricultural ecological efficiency increased to 0.5453. The impact of rural science and technology investments on agricultural ecological efficiency presented as an inverted U-shape, and the scale of agricultural science and technology investments had a significant spillover effect on agricultural ecological efficiency. The panel threshold regression showed that the threshold effect of agricultural science and technology investments in the eastern, middle, and western regions in China differed, and that in the eastern region had a positive promoting effect. The positive effect of agricultural science and technology input on agricultural ecological efficiency in the middle region was not as stable as that in the eastern region. The input of agricultural science and technology in the western region harmed agricultural ecological efficiency. The scientific and technological input in the agricultural development of the middle and western regions of China should consider economic and ecological efficiency. Therefore, China should vigorously promote the green and efficient technology mode, actively replace chemical fertilizers with organic fertilizers, accelerate the implementation of scientific fertilizer application techniques, pay close attention on demonstration, fertilizer reduction and fertilization efficiency, and improve agricultural ecological efficiency.