MicroRNAs (miRNAs) play vital roles in down-regulating gene expression at the post-transcriptional level. A set of 24 UV-B stress-responsive miRNAs (13 up-regulated and 11 down-regulated) was identified in Populus tremula plantlet by expression profiling with our in-house miRNA filter array. Six of the UV-B-responsive miRNA and their corresponding target genes were verified for their expressions by RNA blotting and quantitative reverse transcription PCR (qRT-PCR), respectively. The predicted target genes for these miRNAs encode diverse proteins including transcription factors and phytohormone signal-related proteins. Promoter analysis of the UV-B-responsive miRNAs revealed the presence of many light-relevant cis-elements. However, these cis-elements were not necessarily specific to the promoters of UV-responsive miRNAs, indicating that other machinery may be involved in the regulation of UV-responsive miRNAs. Finally, a model was developed to describe the potential regulatory networks mediated by the UV-B-responsive miRNAs in P. tremula. These results provide new insights into the understanding of miRNAs as ubiquitous regulators in plant response to UV-B and other stresses.