Soybean [Glycine max (L.) Merr.] rotation has been shown to enhance grain sorghum [Sorghum bicolor (L.) Moench] growth and yield due in part to N contribution. Sorghum grain and stover yield, yield components, soil water and soil NO3–N were measured in a long‐term rotation study in 2003 and 2004 on a Sharpsburg silty clay loam (fine, smectitic, mesic Typic Argiudoll). The objectives were to separate biologically fixed N from other rotation effects on sorghum grain and stover yields, and to relate yield to yield components, soil NO3–N and water contents. The cropping sequences were continuous grain sorghum, and sorghum rotated with non‐nodulating or nodulating soybean. Soil amendment treatments consisted of control (zero), manure (17–25 Mg dry matter ha−1 yr−1), and N (41 kg ha−1 for soybean and 84 kg ha−1 yr−1 for sorghum). Cropping sequence × soil amendment interaction effects were found for most parameters measured. High soil NO3–N following soybean rotation and from amendment application promoted plant growth leading to low soil water content at anthesis, and increased kernel weight, grain and stover yield. Rotation with non‐nodulating soybean without soil amendment increased grain yield by 2.6 to 3.0 Mg ha−1 and stover yield by 1.5 to 1.8 Mg ha−1 over continuous sorghum without soil amendment. Rotation with nodulating soybean further increased grain yield by 1.7 to 1.8 Mg ha−1 and stover yield by 0.6 to 0.9 Mg ha−1. Biologically fixed N effects accounted for only 35 to 41% of enhanced sorghum yield due to crop rotation with soybean. Soil NO3–N during vegetative growth, plant height, soil water content at anthesis and kernel weight were the most important parameters related to sorghum grain yield across cropping sequences and soil amendments.