•Potentially mineralizable N (N0) and labile C and N organic fractions were significantly correlated.•Labile N fractions were higher in NT than in CT at all three depths.•The N0 proved to be positively influenced by the quantity and quality of POM-C.•Crop N uptake was not related to N0 for any tillage system and depth. Tillage systems strongly affect nitrogen (N) mineralization. However, there is still only limited information on the relationship between N in labile soil organic matter (SOM) fractions and crop N uptake under different tillage systems in areas with poor water availability. This study discusses the long-term effect of two tillage systems on i) the N-content in labile organic matter fractions and their relationship with the N mineralization potential at three depths (0–5; 0–10 and 0–20cm), ii) the factors that affect the N mineralization potential, and iii) the relationship between potentially mineralizable N (N0) and crop N uptake in a semi-humid climate. In a long-term experiment, a Typic Argiudoll was sampled under two contrasting tillage systems: no-tillage (NT) and conventional tillage (CT). The soil sampling was performed over four years of the crop sequence (2003, 2009, 2010 and 2011) when the plots were sown with winter wheat (Triticum aestivum L.). They were analyzed for N0 in the form of anaerobic N, soil organic nitrogen (SON), physically separated SOM fractions and crop N uptake. Higher values of SON and labile soil N fractions were observed under NT at all three depths. Significant differences in N0 were found between the tillage systems, with greater values under NT. Significant (P<0.05) and positive correlations between N0 and fine particulate organic carbon (fPOM-C) (r≥0.66) were found in CT and in NT at the three depths, whereas highly significant (P<0.001) and negative relationships between N0 and fine particulate organic N (fPOM-N) (r≥−0.83) were found under both tillage systems at 0–5 and 0–10cm. The most pronounced difference in these relationships between tillage systems was observed at the 0–5cm soil depth. Significant correlations of N0 with residue input from previous crops and the fallow period were observed under both tillage systems and for all three depths. Regarding the relationships between N0 and wheat N uptake, no significant correlations were found for any tillage system or depth. Soil organic N fractions were shown to be strongly influenced by the residue input from the previous crop and by variable weather conditions during the fallow period. The higher content of SON fractions under NT was associated with a higher N mineralization potential, however, it did not result in increased N availability and N uptake by wheat, because of climatic conditions during the crop growing season.