The expression profiles of miRNAs in thymus tissues from mice of different age have been demonstrated in our previous study. After an integrated analysis of the miRNA expression profiles, we demonstrated that the expression of miR-181a-5p was significantly decreased in thymic epithelial cells (TECs) from 10- to 19-month-old mice when compared with that in TECs from 1-month-old mice by quantitative reverse transcriptase polymerase chain reaction. We hypothesized that miR-181a-5p in TECs might be associated with the age-related thymus involution through regulating some genes or signaling pathway. To test this hypothesis, the mouse medullary thymic epithelial cells (MTEC1) were used. Transfection with miR-181a-5p mimic promoted the proliferation of MTEC1 cells, but did not affect apoptosis. The effect was reversed when the expression of miR-181a-5p was suppressed in MTEC1 cells. Furthermore, the transforming growth factor beta receptor I (Tgfbr1) was confirmed as a direct target of miR-181a-5p by luciferase assay. Moreover, it was found that overexpression of miR-181a-5p down-regulated the phosphorylation of Smad3 and blocked the activation of the transforming growth factor beta signaling. Nevertheless, an inversely correlation was observed between the expression of Tgfbr1 and miR-181a-5p in TECs derived from mice of different age. Collectively, we provide evidence that miR-181a-5p may be an important endogenous regulator in the proliferation of TECs, and the expression levels of miR-181a-5p in TECs may be associated with the age-related thymus involution.