The aim of this article is to study the effect of tumor necrosis factor alpha (TNF-α) on the expression of matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs) in keratoconus fibroblasts in vitro. Normal cornea and keratoconus fibroblasts were extracted using enzyme digestion method and were cultured in the medium containing TNF-α (0, 10 and 100 ng/mL). The expression of MMPs proteins in the supernatant of corneal fibroblasts and the expression of TIMPs in the normal cornea and keratoconus fibroblasts were detected by Western blot and real-time quantitative polymerase chain reaction respectively. The active form of MMP1 could be detected in the supernatant of keratoconus fibroblasts and upregulated by TNF-α. TNF-α could increase the protein expression of MMP2, MMP3, MMP9 in the supernatant of keratoconus fibroblasts and decrease the gene expression of TIMP1, TIMP2 in keratoconus fibroblasts. The increased MMPs and the decreased TIMPs can increase the degradation of the extracellular matrix. TNF-α may play an important role in the occurrence and development of keratoconus by regulating the expression of MMPs/TIMPs.