Soil health changes induced by prairie reconstruction (cultivated fields to tallgrass prairie) were assessed in Central Missouri within sites representing a chronosequence of 0, 2, 3, 4, 6, 9, 10, 11, 12, and 13-yr postreconstruction. In addition, a nearby remnant native prairie, two long-term reconstructed prairies (-25 and -57-yr post-reconstruction), and a biofuel prairie 9-yr post-reconstruction were evaluated for comparative purposes. From 0 to 8-yr, prairie reconstruction increased soil aggregation, total soil organic carbon (SOC), total nitrogen (TN), active C and N (permanganate oxidizable C and total protein), and mineralizable C and N (soil respiration and potentially mineralizable nitrogen), becoming more similar to levels in the remnant prairie. Further, four enzymes involved in the cycling of C (13-glucosidase), N (13-glucosaminidase), P (acid phosphatase), and S (arylsulfatase) demonstrated amplified activities within samples collected to a depth of 15-cm. Over time, the ratios of active C to SOC and active N to TN declined, reflecting the conversion of active C/N pools into more stable C/N pools due to continued organic inputs and increased microbial activity. In contrast, from 8- to 13-yr post-reconstruction, the number of these same soil health indicators declined, which may be attributed to historical land use, the improvement of prairie reconstruction and management strategies, and ecological processes related to succession. Overall, prairie reconstruction holds great potential for soil health restoration in degraded agricultural landscapes, and further study is needed to understand how historical land use and prairie reconstruction practices affect soil health and ecological resilience.