Transforming growth factor-β1 (TGF-β1) expression in smooth muscle cells may play an important role in the pathogenesis of asthma. However, mechanisms that are involved in the regulation of TGF-β1 gene expression in human airway smooth muscle cells (HASMCs) remain elusive. Here, we show that mechanical stretch of HASMCs augmented TGF-β1 expression through a de novo RNA synthesis mechanism. Luciferase reporter assays revealed that stretch-induced TGF-β1 expression was mediated through the enhanced activation of TGF-β1 promoter. Interestingly, selective inhibitors of PTK, PI3K, or MEK1/2 attenuated TGF-β1 expression through blocking ERK1/2 phosphorylation and TGF-β1 promoter activity in response to stretch. In addition, stretch rapidly and transiently augmented GTP-bound RhoA and Rac1 but not Cdc42 GTPase. Either blockade of RhoA GTPase using C3 transferase, ROCK1/2 using Y27632, or knockdown of endogenous RhoA using RhoA siRNA attenuated stretch-induced TGF-β1 expression through the inhibition of ERK1/2 phosphorylation. Moreover, stretch augmented DNA binding activity of AP-1 in a time-dependent manner. Either treatment of HASMCs with the inhibitors of RhoA, ROCK1/2, PTK, PI3K, MEK1/2, or AP-1 or transfection of HASMCs with AP-1 decoy oligonucleotide attenuated stretch-induced TGF-β1 expression through repressing the DNA binding activity of AP-1. Site-directed mutagenesis demonstrated that two AP-1 binding sites in the TGF-β1 promoter region are responsible for stretch-induced TGF-β1 expression. Overall, in HASMCs, mechanical stretch plays an important role in TGF-β1 gene upregulation through a stretch-induced signaling pathway, which could be a potential therapeutic intervention for TGF-β1-induced pathogenesis in asthma.