Objective To explore the combined effects of mechanical stretch and interleukin-1β (IL-1β) on gene expression of extracellular matrix in rabbit corneal fibroblasts. Methods Isolated rabbit corneal fibroblasts were subjected to 15% equibiaxial stretch at frequency of 0.1 Hz for 12 h, 24 h and 36 h, respectively, in presence of IL-1β. The gene expressions of matrix metalloproteinases (MMPs), tissue inhibitor of metalloproteinases 1 (TIMP-1) and collagen type I alpha 1 (Collagen Iα1) were detected by real-time quantitative PCR. Results The mRNA levels of MMP-1, MMP-3 and MMP-9 could be up-regulated by IL-1β alone. However, MMP-1 and MMP-3 mRNA levels decreased with time, while MMP-9, TIMP-1 and collagen Iα1 increased with time. Compared with corresponding IL-1β treatment with mechanical stretch groups, the mRNA levels of MMP-1, MMP-3 and MMP-9 were increased and the mRNA levels of TIMP-1 and collagen Iα1 were decreased in a time-dependent manner. The mRNA level of Collagen Iα1 was decreased by loading mechanical stretch alone, and would further decrease time-dependently in combination with IL-1β treatment. Conclusions Mechanical stretch combined with IL-1β may facilitate the corneal tissue damage, thereby contribute to the development of keratectasia.