MicroRNAs (miRNAs) are important regulators of cell proliferation, differentiation and function. Mechanical strain is an essential factor for osteoblast proliferation and differentiation. A previous study revealed that a physiological mechanical tensile strain of 2500 microstrain (με) at 0.5 Hz applied once a day for 1 h over 3 consecutive days promoted osteoblast differentiation. However, the mechanoresponsive miRNAs of these osteoblasts were not identified. In this study, we applied the same mechanical tensile strain to in vitro cultivated mouse MC3T3-E1 pre-osteoblasts and identified the mechanoresponsive miRNAs. Using miRNA microarray and qRT-PCR assays, the expression patterns of miRNAs were evaluated and 5 of them were found to be significantly different between the mechanical loading group and the control group: miR-3077-5p, 3090-5p and 3103-5p were significantly upregulated and miR-466i-3p and 466h-3p were downregulated. Bioinformatics analysis revealed possible target genes for these differentially expressed miRNAs. Some target genes correlated with osteoblast differentiation. These findings indicated that the mechanical strain changed the expression levels of these miRNAs. This might be a potential regulator of osteoblast differentiation and responses to mechanical strain.