[Background] The endophytic fungus Piriformospora indica colonizes plants and significantly promotes plant growth and development. miRNAs have been demonstrated to play a regulatory role in plant growth and development. [Objective] To reveal the response of miRNA to P. indica colonization and its regulatory role in the growth and development of barley. [Methods] After extraction of total barley RNA, high-throughput sequencing was performed, followed by sequence alignment and data mining. Then, qPCR was conducted to determine the levels of miRNA and target gene expression. High performance liquid chromatography was employed to measure the levels of growth hormones in barley. [Results] P. indica significantly promoted the growth of barley. The whole transcriptome sequencing results showed that the barley infected by P. indica for three days had 11 miRNAs up-regulated and 7 miRNAs down-regulated compared with the blank control group; the barley infected by P. indica for seven days had 11 miRNAs up-regulated and 13 miRNAs down-regulated compared with the blank control group; the barley infected by P. indica for seven days had 3 miRNAs up-regulated and 6 miRNAs down-regulated compared with the barley infected by P. indica for three days. The GO functional annotation and KEGG pathway enrichment analysis showed that predicted target genes of these differentially expressed miRNAs were mainly involved in transcription, cell division, auxin signal perception and transduction, photosynthesis, and response to hormone stimulus. These pathways were associated with barley growth, which suggested that miRNAs responded positively to the colonization of P. indica. In addition, the metabolites of the regulatory pathways involving the differentially expressed miRNAs changed. [Conclusion] This study explored the regulatory role of miRNA in barley growth and development, providing a new research direction for deciphering the growth promotion mechanism of P. indica colonization.