Improved understanding of the effects of broiler litter application to row crops as a source of nutrients on soil properties is required to predict the outcome of litter applications and prevent undesirable environmental impacts. This study was conducted on two separate commercial farms representing a no-till system at Coffeeville and a conventional-tillage system at Cruger in Mississippi to evaluate soil chemical, physical, and biological properties in response to 3 yr of broiler litter application with or without supplemental N. In each location, the experimental design was a randomized complete block with six treatments replicated four times. Treatments included broiler litter rates of 0, 4.5, and 6.7 Mg ha−1 in some combination with 0, 34, and 67 kg N ha−1 Commercial N-P-K fertilizer at the recommended rate was included. At the Coffeeville location, changes in soil characters increased with increasing broiler litter applications, and at the rate of 6.7 Mg ha−1, soil pH, P, total N, total C, microbial biomass C, and soil aggregate stability significantly increased by 22, 20, 18, 20, 28, and 34%, respectively, as compared with commercial fertilizer treatment at the recommended rate. At the Cruger location, changes in soil character were less responsive to treatment applications. For the same broiler litter rate at Coffeeville and Cruger, no significant difference in the changes of soil character was obtained between a combination of broiler litter with supplemental N and broiler litter alone treatments. At both locations, broiler litter at rates >4.5 Mg ha−1 with 67 kg ha−1 supplemental N did not increased cotton lint yield and exceeded the crop N use potential as evidenced by increasing soil profile NO3–N.