Technical Abstract: Understanding long-term management effects on soil properties is necessary to determine the relative sustainability of cropping systems. Soil physical, chemical, and biological properties were measured in a long-term cropping system study in the Western Corn Belt. Properties were evaluated after 16 years in four crop sequences [continuous corn (CC), corn-soybean (C-SB), corn-oat+clover-grain sorghum-soybean (C-OCL-SG-SB), and corn-soybean-grain sorghum-oat+clover (C-SB-SG-OCL)] each at three N fertilization rates (ZERO, LOW, and HIGH) to a soil depth of 30.5 cm on a Sharpsburg silty clay loam (fine, smectitic, mesic Typic Argiudoll). Nitrogen fertilization had a greater impact on soil properties than crop sequence, with management effects most pronounced at 0-7.6 cm. Increased N-rate resulted in higher organic C, total N, and particulate organic matter (POM). Organic C, in particular, was 1.4 Mg ha-1 higher in the HIGH HN-rate treatment as compared to the ZERO N-rate treatment. Increased N-rate, however, resulted in lower soil pH levels and reduced microbial biomass by about 20% between the HIGH and ZERO N-rate treatments. Four-year crop sequences had more potentially mineralizable N (55 kg ha-1 for four-year sequences vs. 46 kg ha-1 for average of CC and C-SB) and higher percentages of POM present as soil organic matter (17.1% for the C-SB-SG-OCL sequence vs. 13.9% for other sequences). Results from this evaluation indicate 4-yr crop sequences promoted a balance of soil characteristics that should enhance soil functions over time.