Iron (Fe) deficiency in calcareous soils of the Loess Plateau of China is a wide spread issue and primarily affects agricultural production due to the relatively higher soil pH and carbonate content. Understanding the relationships between Fe distribution in soil fractions, Fe availability, and their responses to cropping and fertilization could provide essential information for assessing Fe availability in soils and managing soils to improve Fe availability. A long-term field experiment was established in 1984 in a split-plot design using cropping systems as main plots and fertilizer treatments as subplots on a farmland in the Loess Plateau. The cropping systems included fallow, continuous wheat cropping, continuous alfalfa cropping, continuous maize cropping, and a rotation system that included a legume. Various fertilization treatments using chemical and/or manure fertilizers were included in each cropping system. Soil samples were collected from 0–10 and 20–40 cm depths in 2012. Long-term planting of crops significantly increased the concentrations of available Fe in the soils. The largest increase was observed in the continuous alfalfa cropping system. Long-term cropping significantly increased the concentrations of Fe associated with carbonates and organic matter, but decreased the concentration of Fe associated with minerals in the soils. The effect of fertilization on the distribution of Fe in the soil fractions varied with cropping system and soil depth. The fertilization treatment with manure generally increased the concentrations of Fe associated with the soil fractions. Long-term cropping and fertilization in the highland farmland significantly affected the availability of Fe and the distribution of Fe fractions in the soil.