Our previous research found that Q-switched 1064-nm Nd: YAG laser (1064-QSNYL) induces skin collagen synthesis by activating TGFβ1/Smad3/p38MAPKs pathway. Moreover, a lot of studies shown that MicroRNAs (miRNAs) are contribute to regulate collagen synthesis and skin barrier. Therefore, we intend to explore the mechanism of 1064-QSNYL on collagen synthesis and skin barrier through miRNAs. We predicted the upstream miRNAs of TGFβ1 by bioinformatics databases, and verified them through dual-luciferase reporter genes and Western blotting. The expression of Collagen, skin barrier related protein K10 and Filaggrin, TIMP-1 and MMP-2 were detected by RT-qPCR and Western blotting, respectively. Moreover, we detected moisture content, elasticity value, TEWL value, SOD vitality and Hydroxyproline content to evaluate skin barrier of mice. H&E staining to observe the changed of dermis thickness and inflammation and infiltration of mice skin. The results shown that TGFβ1 was target gene of miR-663a. Moreover, we found that 1064-QSNYL activated TGFβ1/smad3/p38MAPK pathway by downregulating the expression of miR-663a in HaCaT, HDF cells and mice, thereby promoting expression of Collagen I, Collagen IV, TIMP-1, K10 and Filaggrin and inhibiting MMP-2. Furthermore, 1064-QSNYL contributed to moisture content, elasticity, SOD vitality and Hydroxyproline content via miR-663a to activate TGFβ1/smad3/p38MAPK pathway. In summary, this study found for the first time that 1064-QSNYL contributed to collagen synthesis and skin repair via miR-663a to regulate TGFβ1/smad3/p38MAPK pathway, thereby achieving skin rejuvenation. This article is protected by copyright. All rights reserved.