Understanding wheat straw nitrogen (N) transformations in soil depending on tillage practices is necessary to increase the efficient N use and to protect dryland farming environments. Labile organic N pools are crucial for N mineralization but are less investigated because of their fast dynamics. The objectives of this study were to determine the effects of winter wheat straw N (15N-labelled) on soil labile organic N transformations under long-term (27 years) no-tillage and conventional tillage. Four treatments were established under controlled conditions: no-tillage without straw (NoTill); no-tillage with 15N-labelled wheat straw (NoTill+Straw); conventional tillage without straw (ConvTill); and conventional tillage with 15N-labelled straw (ConvTill+Straw). Straw application increased the particulate organic matter N (POMN) and microbial biomass N (MBN) contents, especially in NoTill+Straw, but decreased the dissolved organic N (DON) content. This reflects intensive microbial DON immobilization at the background of plant residues with a high C/N ratio. The 40% to 80% increase in the NH4+ content after straw addition reflects fast straw and soil organic matter (SOM) mineralization, while NO3− declined by 60% to 93% over 56 days, especially in ConvTill soil. 15N recovered in POMN after 14 d was greatest in NoTill+Straw and ConvTill+Straw, reaching 20 and 18 mg 15N kg−1, respectively. Overall, the straw N recovered as NO3− was 72% and 48% greater than that in NH4+ in ConvTill+Straw and NoTill+Straw, respectively, reflecting accelerated SOM mineralization and N mining in the presence of straw. The straw N recovered as POMN was greater through the incubation period (56 days) than that in DON and MBN, especially in NoTill+Straw, indicating that the no-tillage practice reduced straw mineralization. 15N in DON and 15N in NO3− were higher in NoTill+Straw than in ConvTill+Straw, implying that N limitation was alleviated to a certain extent under no-tillage conditions. In conclusion, wheat straw N remained mainly in POMN after 56 days, especially under no-till conditions, and only a minor part was incorporated into DON and microbial biomass. This provides a theoretical basis for straw N use efficiency in agroecosystems.