ABSTRACT: The objective of this experiment was to study the effect of cotton residues incorporation on soil properties, soil organic nitrogen (N) fractions, and N-mineralizing enzyme (protease, and urease) activity in the 0-40 cm soil layer in the long-term continuous cotton field. In this experiment, seven treatments, including cotton residues incorporation for 5, 10, 15 and 20 years (marked as 5a, 10a, 15a, and 20a) and continuous cropping for 5, 10 and 20 years (marked as CK5, CK10 and CK20) were conducted. The results showed that the soil organic carbon (C) and N increased gradually with the increase in the duration of continuous cropping with cotton residues incorporation. Compared with CK20, the 20a treatments reduced the content of amino acid N (AAN), ammonium N (AN), amino sugar N (ASN), hydrolysable unidentified N (HUN), and acid insoluble N (AIN) significantly by 48.6, 32.2, 96.9, 48.3, and 38.7%, respectively (p < 0.05). The activity of protease and urease in 20a treatments significantly increased by 53.4 and 53.1% respectively as compared to CK20 (p < 0.05). Soil organic C and N-mineralizing enzyme activity decreased with the increase in cropping duration in the absence of cotton residues incorporation, while the organic N increased slightly. In conclusion, cotton residues returning can increase the storage of soil organic C and N in long-term continuous cropping cotton field, and improve the soil quality and soil fertility of continuous cropping cotton field.