The effects of long-term (16 years) fertilization on the diversity and community structure of soil ammonia-oxidizing gene (amoA) and hydroxylamine-oxidizing gene (hao) in paddy soil were evaluated using the methods of polymerase chain reaction, cloning and sequencing. The soil samples were collected from the treatments of NPK (CK) and NPK plus rice straw (SR) of the long-term field fertilization experiment in Taoyuan Agro-ecological Experimental Station. The Shannon Indices showed that the diversity of amoA and hao in SR treatment was lower than that in CK, and LUBSHUFF statistical analyses demonstrated that the sequence compositions of both amoA and huo libraries were significantly different between CK and SR. The phylogenetic trees indicated that some clusters appeared in SR treatment but were not detected in CK treatment. As to amoA, only Nitrosospira besides the uncultured amoA sequences were cloned from the two treatments, while no Nitrosomonas species were detected. As to hao, the strains from Silicibacter and Methylococcus were dominant in CK, while in SR the strains from Nitrosospira and Nitrosomonas were dominant. Sum up, the long-term rice straw application has caused a remarkable impact on the diversity and community structure on Nitrosobacteria.