MicroRNAs (miRNAs) are 20–24 nucleotide (nt) non-coding regulatory RNAs which play critical roles in plant growth and development. miRNA-encoding genes, which are transcribed by RNA polymerase II, are involved in a variety of processes, including developmental and morphogenesis systems, and hormone and stress responses. To investigate miRNA responses to tissue culture conditions, quantitative reverse transcription–polymerase chain reaction (qRT-PCR) was used to detect differences in miRNA expression between in vitro micropropagated strawberry plants and transplanted micropropagated strawberry plants. Four miRNAs were differentially expressed between them, including one up-regulated gene (miR156) and three down-regulated genes (miR164, miR172 and miR390). The ratios of miRNA expression levels in in vitro micropropagated strawberry plants to micropropagated plants transplanted into soil in greenhouse for 4 months for miR156, miR164, miR172 and miR390 were 6.757, 0.046, 0.035 and 0.050, respectively. The ratio of miR156 expression levels in micropropagated plants transplanted into soil for 5 months to levels in the conventionally propagating runner plants was 3.785. miR156 was expressed highly and was strikingly inversely proportional to the expressions of its target gene SPL9 and miR172 in in vitro micropropagated strawberry plants. We speculate that high expression of miR156 is the main reason for rejuvenation in micropropagated plants.