It remains unclear how the expression of microRNAs (miRNAs) in human periodontal ligament stem cells (PDLSCs) might respond to mechanical stretch. To investigate specific miRNA expression in stretched PDLSCs, we used a Flexcell® FX-5000™ tension system to achieve external mechanical stimulation. Then, a custom-designed microarray assay was performed to investigate and describe the genome-wide differential expression of miRNAs in normal and stretched PDLSCs. Finally, we implemented integrative miRNA target prediction and network analysis approaches to construct an interaction network of the key miRNAs and their putative targets. We found that stretching induced morphological changes and increased alkaline phosphatase (ALP) activity, runt-related transcription factor 2 (RUNX2), osteocalcin (OCN), and bone sialoprotein (BSP) expression in PDLSCs. The microarray data showed that 53 miRNAs were differentially expressed with stretching. With an interaction network, we examined the connections between 10 selected key miRNAs and their putative target genes, which were related to mechanical force. The results from the interaction network provided a basis for postulating the functional roles of miRNAs in PDLSCs.