We previously demonstrated that collagen metabolism affects scleral mechanical properties and scleral remodeling. Scleral remodeling changes the mechanical strain on sclera and scleral fibroblasts. We postulated that mechanical strain changes affect collagen metabolism in scleral fibroblasts. To understand the differences in collagen metabolism in scleral fibroblasts related to mechanical strain changes, scleral fibroblasts were isolated and cultured under different mechanical strains using the FX-4000 system or were treated with the TGF-β1 and TGFBR1 inhibitor LY364947. The collagen metabolism-related gene expression levels were detected. The results showed that the appropriate (lower) mechanical strain improved collagen synthesis and reduced collagen decomposition. In contrast, higher mechanical strain reduced collagen synthesis and enhanced collagen decomposition, especially a sustained higher strain. Furthermore, the effect of a transitory higher strain was recoverable, and collagen metabolism in scleral fibroblasts was regulated by TGF-β1. These results suggested that mechanical strain mediates TGF-β1 expression to regulate collagen metabolism in scleral fibroblasts, thereby affect scleral tissue remodeling.