The objective of this paper was to explore the impact of environmental regulation and economic agglomeration on regional ecological efficiency in China. First, this paper adds energy factor and pollutant emission output to the production function and proves by the derivation of the mathematical model that environmental regulation and economic agglomeration can affect eco-efficiency. Second, based on the panel data of 30 province-level regions in China from 2003 to 2017, the dynamic spatial panel Durbin model and the mediating effect test model are used to empirically analyze the relationship between the three. The results show that economic agglomeration and eco-efficiency present a typical N-shaped curve relationship. Environmental regulation can enhance eco-efficiency and economic agglomeration, and economic agglomeration can be a mediator of environmental regulation to affect eco-efficiency. The joint implementation of environmental regulation and agglomeration economic is helpful to the improvement of ecological efficiency. Both eco-efficiency and economic agglomeration have significant time dependence and spatial spillover effects. From a regional perspective, the marginal role of environmental regulation in promoting eco-efficiency in the central and western regions is greater than that in the eastern regions. The impact of economic agglomeration on eco-efficiency still shows significant N-type characteristics in the central region, while it shows an inverted N-type characteristic in the eastern region, but it is not significant in the western region.