Abstract: Yield gap reflects the increasing room for grain production and reducing the gap is an important way to increase grain yield. Analyzing the constraint factors of the yield gap can provide guidance for reducing yield gap and increasing grain output. Based on the statistical data of cities in Jiangsu Province from 2000 to 2018 and applying the boundary line analysis method, this paper analyzed the contribution rates and the optimal values of constraint factors. Results show that: 1) the average yield gap of grain production accounts for 23.47% and 64.05% of the sample’s highest yield and the average yield, respectively, indicating that there is a large room for improvement in grain yield in Jiangsu Province; 2) the key constraint factors for the yield gap include the multiple crop index, labor, and grain yields in southern, central, and northern Jiangsu; 3) the grain output is the highest when the multiple crop index is 1.6, the total mechanical power is 5.3 kW/hm2, and the fertilization intensity is 487.64 kg/hm2, which are the optimal value of factors of grain production. In addition, the fertilization intensity exceeds the warning line of fertilization, indicating that the agricultural fertilization efficiency is low in Jiangsu Province; and 4) it can increase grain yield by 30.3%, 17.73%, and 21.45%, while reducing the demand for farmland by 8.8%, 13.38% and 9.02% by converting yield gap in southern, central and northern Jiangsu, respectively. Therefore, to reduce the gap in grain output, to promote sustainable grain production, and to ensure grain security in Jiangsu Province, this paper provides the following policy suggestions: increasing the scale of multiple cropping of farmland, improving agricultural production conditions, strengthening the guidance of proper fertilizer application, and improving the level of mechanization.