Staphylococcus aureus is a common bacterial isolate from cases of microbial keratitis. The virulence factors that contribute to its pathogenicity during this disease have not been fully resolved. The aim of the current study was to examine the effects of the extracellular protease Staphopain A on corneal virulence. Two strains were used, one Staph 38 that gives a high pathology score during keratitis and a less virulent strain ATCC 8325-4. The effect of inhibition of Staphopain by general or specific protease inhibitors on adhesion of strains to fibronectin-coated glass or PMMA was determined. This was followed by an analysis of the effect of Staphopain A on the ability of the bacteria to adhere to and invade corneal epithelial cells. Finally, the effect of inhibiting Staphopain A on pathogenesis in a mouse model of keratitis was studied. Staphopain A increased the adhesion of strains to fibronectin–coated substrata and inhibition of Staphopain A reduced adhesion. The inhibition of Staphopain A by staphostatin A significantly decreased both association with and invasion into human corneal epithelial cells by 15-fold for strain Saur38. Inhibition of Staphopain A significantly reduced the pathology associated with S. aureus keratitis, reducing the infecting numbers of bacteria from 1.8x105 to <1x104 cells/cornea (p ≤ 0.001), significantly reducing the corneal pathology score (p ≤ 0.038) and reducing the numbers of infiltrating PMNs. This study shows that Staphopain increases adhesion and invasion of corneal cells due to increasing fibronectin binding and its inhibition has a significant impact on pathogenicity of S. aureus during keratitis.