The paper reports for the first time, in poplar woody root, the expression of five mechanically-responsive miRNAs. The observed highly complex expression pattern of these miRNAs in the bent root suggest that their expression is not only regulated by tension and compression forces highlighting their role in several important processes, i.e., lateral root formation, lignin deposition, and response to bending stress. Mechanical stress is one of the major abiotic stresses significantly affecting plant stability, growth, survival, and reproduction. Plants have developed complex machineries to detect mechanical perturbations and to improve their anchorage. MicroRNAs (miRNAs), small non-coding RNAs (18-24 nucleotides long), have been shown to regulate various stress-responsive genes, proteins and transcription factors, and play a crucial role in counteracting adverse conditions. Several mechanical stress-responsive miRNAs have been identified in the stem of Populus trichocarpa plants subjected to bending stress. However, despite the pivotal role of woody roots in plant anchorage, molecular mechanisms regulating poplar woody root responses to mechanical stress have still been little investigated. In the present paper, we investigate the spatial and temporal expression pattern of five mechanically-responsive miRNAs in three regions of bent poplar woody taproot and unstressed controls by quantitative RT-PCR analysis. Alignment of the cloned and sequenced amplified fragments confirmed that their nucleotide sequences are homologous to the mechanically-responsive miRNAs identified in bent poplar stem. Computational analysis identified putative target genes for each miRNA in the poplar genome. Additional miRNA target sites were found in several mechanical stress-related factors previously identified in poplar root and a subset of these was further analyzed for expression at the mRNA or protein level. Integrating the results of miRNAs expression patterns and target gene functions with our previous morphological and proteomic data, we concluded that the five miRNAs play crucial regulatory roles in reaction woody formation and lateral root development in mechanically-stressed poplar taproot.