The effect of long-term (20 yr) fertilizer application on soil nitrogen (N) transformation in paddy soils was studied at three sites (Xinhua, Ningxiang, and Taojiang) in Hunan province, China. Four fertilization practices were chosen: chemical fertilizers (NPK), chemical fertilizers plus a medium or high amount of pig manure (MM NPK), chemical fertilizers plus a high amount of pig manure (HM NPK), and chemical fertilizers plus straw incorporated (Str. NPK). A treatment with no fertilization was included as a control (CK). Ten week aerobic incubations were conducted to determine N potential mineralization and nitrification. Application of organic plus chemical fertilizer increased soil organic carbon, total nitrogen, and microbial biomass carbon (C_mic) and nitrogen (N_mic), whereas the response of C_mic/N_mic ratio to fertilizer application varied among sites. Across all sites, the HM NPK treatments had the highest potentially mineralizable N and maximal nitrification rate, and the CK had the lowest. The MM NPK, Str. NPK, and NPK treatments had lesser effects on mineralizable N and nitrification. Results indicated that chemical fertilizer along with a high rate of manure application is an effective method to improve available soil N by increasing the N mineralization rate. However, higher N nitrification was also induced by manure application, which may lead to increased N losses, and also should be considered in practical applications.