MAIN CONCLUSION: 87 miRNAs responding to low nitrogen stress in poplar roots were identified by miRNA-seq, and their target genes were predicted. Additionally, several key miRNA-mRNA modules were summarized. ASBTRACT: Nitrogen (N) is an essential nutrient for plants, and low nitrogen (LN) availability can constrain plant growth and development. MicroRNAs (miRNAs) play an important role in plant response to nutrient stress as a regulatory factor. However, studies on the function of poplar miRNAs under LN stress are limited. In this study, we investigated the potential role of miRNA in poplar roots under LN stress using miRNA-seq. 305 conserved miRNAs belonging to 48 miRNA families were identified, and 15 novel miRNAs were predicted. Among these, 83 known miRNAs from 21 families and 4 novel miRNAs were confirmed as differential expressed miRNAs (DEMs) following LN stress treatment at 6, 9, 24, 72, 240, and 504 h compared to 0 h. Functional annotation analysis indicated that an array of miRNAs, including miR160, miR172, and miR166, should be involved in LN stress. TargetFinder and psRobot predicted that 52 of these miRNAs target 248 genes, resulting in 319 miRNA targeting pairs. Degradome sequencing further revealed that these 52 miRNAs targeted 457 genes, with 358 miRNA-target pairs. Gene annotation of target genes indicated that AP2, ARF, HD-ZIP, and other genes might respond to LN stress by regulating root growth and development. These findings provide valuable insights into miRNA functions and establish a framework for further investigating miRNA-mediated N signal transduction networks under LN stress. This research may offer new perspectives for genetic engineering to enhance nitrogen use efficiency in forest trees.