The environmental effect of agricultural agglomeration is more complicated than that of other industries, but there is a deficiency of the research on it. This study calculated the level of agricultural agglomeration and the emissions of agricultural non-point pollutants in Chinese prefecture-level cities by using the panel data of these cities on the basis of the theoretical analysis. Then we investigated the spatial and temporal characteristics of agricultural agglomeration and agricultural non-point source pollution and the threshold effect of agricultural agglomeration level on agricultural non-point source pollution based on GIS spatial analysis and econometric model. The main conclusions can be drawn as follows: (1) Agricultural agglomeration and agricultural non-point source pollution of COD, TN and TP show different characteristics in temporal variation, spatial distribution, spatial correlation and spatial trends. (2) Agricultural agglomeration has single threshold effect on COD, TN and TP pollution, and positive correlation with them. When the level of agricultural agglomeration is below the threshold value, it contributes to agricultural pollution significantly, and as the level of agglomeration becomes higher than the threshold value, the situation of rapid pollution will be mitigated. (3) The change from agricultural agglomeration to emissions of three agricultural non-point source pollutants develops in two stages. This change depends on the intensification and restriction of the scale effect, structure effect, technical effect, social effect and cumulative effect on the emissions of agricultural non-point source pollution produced by agricultural agglomeration and it is reflected in the concrete agricultural production behavior. With the increase of agricultural agglomeration, the effect of each effect is obvious and the level of pollution increases significantly, however, the inhibition ability of pollution emission is improved and the external effect of negative environment is weakened at the stage of crossing the threshold. (4) The agricultural economic level and agricultural production conditions aggravate the COD, TN and TP agricultural non-point source pollution to some extent; the structure of animal husbandry industry and environmental regulation have a positive correlation with COD and TP agricultural non-point source pollution; the improvement of producer quality can affect farmers' production behavior and improve the agricultural environment. (5) The effect of agricultural agglomeration on pollution reduction of COD agricultural non-point source pollution is weak and COD agricultural non-point source pollution is more serious and mainly from animal husbandry.