Subsoils have been identified as a potential carbon sink because they typically have low soil organic carbon (SOC) concentrations and high SOC stability. One proposed strategy to increase SOC stocks is to enhance C inputs to the subsoil by increasing crop rotation diversity with deep-rooted perennial crops. Using three long-term field trials in Iowa (study durations of 60, 35, and 12 years), we examined the effects of contrasting cropping systems [maize (Zea mays L.)-soybean (Glycine max (L.) Merr) (= two-year system) vs. maize-soybean-oat (Avena sativa L.)/alfalfa (Medicago sativa L.)-alfalfa or maize-maize-oat/alfalfa-alfalfa (= four-year system)] on above- and below-ground C inputs, as well as the content, biochemical composition, and distribution of SOC among physical fractions differing in stability to 90 cm depth. Average annual total C inputs were similar in the two-year and four-year systems, but the proportion of C delivered belowground was 20–35 % greater in the four-year system. Despite the long duration of these studies, the effect of cropping system on SOC content to 90 cm was inconsistent across trials, ranging from −7 % to +16 % in the four-year relative to the two-year system. At the one site where SOC was significantly greater in the four-year system, the effect of cropping system on SOC content was observed in surface and subsoil layers rather than limited to the subsoil (i.e., below 30 cm). Cropping system had minimal effects on biochemical indicators of plant-derived organic matter or on the proportions of SOC in labile particulate organic matter versus stable mineral-associated organic matter. We conclude that adoption of cropping systems with enhanced belowground C inputs may increase total profile SOC, but the effect is minimal and inconsistent; furthermore, it has minor impact on the vertical distribution, biochemical composition, and stability of SOC in Mollisols of the Midwest U.S.