External mechanical loading on a wound commonly increases fibrosis. Transforming growth factor-β1 (TGF-β1) has been implicated in fibrosis in various models, including the mechanical force model. However, the underlying mechanism is unclear. Our previous experiments suggested that eukaryotic initiation factor 6 (eIF6) acted as a regulator of TGF-β1 expression, and negatively impact on collagen synthesis. Our current results showed that external mechanical stretching significantly increased COL1A1, TGF-β1 and eIF6 expression as well as dermal fibroblasts proliferation, both in vitro and in vivo. eIF6 -deficient (eIF6+/-) cells exhibited significantly higher levels of COL1A1, and these levels increased further with external mechanical stretching, suggesting that mechanical stretching plays a synergistic role in promoting COL1A1 expression in eIF6+/- cells. Inhibition of TGFβR I/II by LY2109761 decreased COL1A1 protein expression in eIF6+/- dermal fibroblasts in a cell stretching model, and attenuated granulation tissue formation in partial thickness wounds of eIF6+/- mice. These data suggest that mechanical stretching has a synergistic role in the expression of COL1A1 in eIF6+/- cells, and is mediated by activation of TGFβRI/II. Taken together, our results indicate that eIF6 may be involved in external mechanical force-mediated murine dermal fibroblast function at least partly through the TGF-β1 pathway.