Comammox Nitrospira represents a groundbreaking discovery in nitrogen cycle research, showcasing its remarking ability for complete ammonia oxidation, which challenges prior conceptions of nitrification. In this study, we examined the response of comammox Nitrospira gene abundance, diversity, and community structure to different rates of pig manure substitution (0 %, 25 %, 50 %, 75 %, and 100 %) in subtropical agroforestry soils. The abundance of ammonia-oxidizing microorganisms was assessed by qPCR, whereas the diversity and structure of comammox Nitrospira were determined by high-throughput sequencing. Our findings revealed that pig manure substitution led to an increase in soil pH, available phosphorus (AP), comammox Nitrospira abundance, and diversity within soils under Cinnamomum camphora coppice planting. Soil pH and AP were the primary factors influencing the diversity and community structure of comammox Nitrospira. Moreover, pig manure substitution significantly influenced the composition of comammox Nitrospira, notably by increasing the relative abundance of clade A.2.1 while reducing that of clade A.2.2. However, pig manure substitution did not exert a significant impact on net nitrification rates, suggesting bacterial relative abundances were more sensitive to manure substitution compared to the underlying biogeochemical processes. Overall, our results offer new insights into the response of comammox Nitrospira to different rates of pig manure substitution in Cinnamomum camphora coppice planting soils, highlighting the pivotal role of soil AP and pH as the key determinants shaping comammox Nitrospira diversity and community structure.