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Abstract Long-term fertilizer application in row crops may influence soil pore characteristics, thereby impacting soil aggregation and structure. Therefore, understanding the influences on soil pore characteristics is useful for adopting suitable conservation practices. However, the impact of cattle manure and inorganic fertilizer application at varied rates on soil pore characteristics in the soil profile at a microscale level remains limited. This study quantifies the impacts of manure and inorganic fertilizer amendments under a corn (Zea mays L.)-soybean (Glycine max L.)-spring wheat (Triticum aestivum) rotation system on soil pore characteristics using the X-ray computed tomography (XCT). Treatments included: low manure (LM; 4.4 and 3.3 Mg ha−1), medium manure (MM; 27.4 and 18.7 Mg ha−1), high manure (HM; 54.8 and 37.4 Mg ha−1), medium fertilizer (MF; 136 kg N ha−1, 49 kg P2O5 ha−1, and 91.5 kg K2O ha−1), high fertilizer (HF; 204 kg N ha−1, 73.5 kg P2O5 ha−1, and 137.3 kg K2O ha−1), and control (CK), respectively, at Brookings (initiated in 2008) and Beresford (2003) in South Dakota. Four intact soil cores were collected from each treatment at 0–10, 10–20, 20–30, and 30–40 cm depths. Results showed that the HM treatment increased the SOC by 8–68% compared to the CK and MF at 0–20 cm at the study sites. Both HM and MM treatments increased the macroporosity and mesoporosity in 0–20 cm soil depths at both study sites. Treatment did not always improve soil pore characteristics below 20 cm soil depth. Additionally, a positive correlation was observed between the XCT-derived macroporosity, total number of macropores, and SOC for all the treatments. Therefore, this study encourages the adoption of the XCT technique in quantifying soil pore characteristics and suggests that long-term medium manure application enhances soil structure as compared to an equivalent inorganic fertilizer application.