Nitrogen (N) is an essential mineral element for plant growth processes, and its availability severely affects the productivity of plants, especially trees. MicroRNAs (miRNAs) are a class of non-coding RNAs approximately 21 nucleotides in length that play important roles in plant growth, development and stress responses. To identify Populus miRNAs and their functions in response to nutrition stress, high-throughput sequencing was performed using Populus tomentosa plantlets treated with or without low concentrations of N. We identified 160 conserved miRNAs, 15 known but non-conserved miRNAs, 2 candidate novel miRNAs and 71 corresponding miRNA*s. Differential expression analysis showed that expression of the 21 conserved miRNA families was significantly altered. Real-time quantitative PCR (qPCR) was used to further validate and analyze the dynamic expression of the identified miRNAs. A total of 218 target genes from the low-N-responsive miRNAs were predicted, and their functions were further annotated in combination with Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. These results suggest that miRNAs play important roles in the response of Populus to low N stress. Furthermore, this study provides the first identification and profiles of N stress-responsive miRNAs from trees.