To study the effects of water and nitrogen regulation on soil organic nitrogen fractions, total nitrogen (TN) and mineral nitrogen (MN) of greenhouse soil (0-30 cm) in fallow period, a tomato field experiment with mulched drip irrigation was conducted with different irrigation lower limits (W1, W2, W3) and nitrogen application rates (N1, N2, N3). The results indicated that greenhouse soil organic nitrogen was mainly in the form of acid-hydrolyzable nitrogen (AHN), and in general, the AHN content was greater than that of non-hydrolyzable nitrogen (NHN). The distributions of soil organic nitrogen fractions under different treatments were significantly different in AHN and NHN. Generally, the order of the contents and distribution proportions of soil organic nitrogen fractions to total N was: amino acid nitrogen (AAN)/ammonia nitrogen (AN)＞unknown nitrogen (UN)＞amino sugar nitrogen (ASN). Except for the ASN, both the contents and distribution proportions of AHN fractions decreased significantly with the increase of soil depth (P＜0.05). Similarly, the contents of TN, MN and total soil organic nitrogen (TSON) also decreased significantly with the increasing soil depth (P＜0.01). Under different treatments, the differences among TN and soil organic nitrogen fractions besides ASN reached a very significant level (P＜0.01). Only AN and AHN had the significant positive correlation with MN (P＜0.01). The results from two-way ANOVAs showed that all the effects of irrigation, nitrogen fertilization and their interaction on TN, MN and TSON reached very significant level (P＜0.01). Therefore, the change of greenhouse soil nitrogen content was closely related to the regulation of water and nitrogen. AN and AAN were the main forms of soil organic nitrogen, the main components of soil available nitrogen, and also the indicator of nitrogen supply potential in greenhouse soil. Considering the soil nitrogen supply potential, the most reasonable pattern of water and nitrogen application was W2N2 in greenhouse soil.