The discovery of comammox Nitrospira challenged the conventional concept of two-step nitrification, which was thought to be performed by two distinct microorganisms, and opened up new avenues to study the relative contribution of different ammonia oxidizers to nitrification. Numerous studies have shown that comammox Nitrospira distributed widely in terrestrial ecosystems, however, their distribution in agricultural soils and how they responded to manure application remained largely unknown. In this study, we collected the soil samples from a long-term field experiment established 18 years ago at Yingtan Red Soil Ecology Experimental Station, Jiangxi, China. We determined the abundances of amoA genes of ammonia-oxidizing bacteria (AOB), archaea (AOA) and comammox Nitrospira using real-time quantitative PCR. The results showed that, compared with none fertilized control, long-term applications of pig manure significantly increased soil organic matter and nutrient contents, which increased with the increasing application rates of pig manure. Moreover, the long-term applications of medium and high rates of pig manure significantly increased soil net nitrification rates by 317% and 416%, respectively. In all the treatments, the abundance of comammox Nitrospira clade B was very low, which could be resulted from non-specific amplification products. Comammox Nitrospira clade A were detected in all the treatments, although their abundances were lower than those of AOA and AOB. The pig manure application significantly increased the abundance of comammox Nitrospira clade A amoA gene, indicating that the comammox Nitrospira in soils were not necessarily oligotrophic. Available phosphorus was a primary factor controlling the abundance of comammox Nitrospira clade A. The Pearson correlation analysis showed that the abundance of comammox Nitrospira clade A was positively correlated with the net nitrification rate, but that of AOA and AOB did not, indicating that comammox Nitrospira clade A might play an important role in nitrification of the soils under long-term applications of pig manure. In conclusion, this study suggests that long-term applications of pig manure increased the abundance of comammox Nitrospira clade A, which might play a critical role in nitrification of the arable Ultisols.