AIM: To observe changes in the content of matrix metalloproteinases (MMPs) in the corneal stroma after corneal cross-linking (CXL) in rabbits, and further explore the corneal pathophysiological process after CXL. METHODS: Forty-two rabbits (42 eyes) were randomly divided into seven groups. One group served as the control group, while the other six groups were treated with CXL. The concentrations of MMPs in corneal stroma were evaluated through parallel reaction monitoring at baseline and 3, 7, 15, 30, 90, and 180d after treatment. RESULTS: The levels of MMP-2 in the corneal stroma of rabbits were 0.76±0.07, 2.78±1.39, 4.12±0.69, 2.00±0.29, 2.00±0.30, 1.22±0.18, and 1.35±0.18 (10-9 mol/g) at baseline and 3, 7, 15, 30, 90, and 180d after treatment, respectively. The contents of tissue inhibitor of metalloproteinase-1 (TIMP-1) were 1.83±0.26, 7.94±0.58, 6.95±2.64, 3.81±0.48, 3.07±0.92, 1.72±0.19, and 1.69±0.74 (10-9 mol/g), respectively. The ratios of MMP-2/TIMP-1 were 0.42±0.33, 0.36±0.20, 0.62±0.10, 0.54±0.15, 0.68±0.13, 0.71±0.10, and 0.68±0.09, respectively. After CXL, the expression of MMP-2 and TIMP-1 in the rabbit corneal stroma was initially increased and subsequently decreased. The levels of MMP-2 remained higher than those recorded at baseline 180d after treatment, but it was not statistically significant. The levels of TIMP-1 returned to baseline levels at 90d after treatment. The ratio of MMP-2/TIMP-1 started to rise from 7d after CXL. It was significantly higher than that calculated at baseline 30-180d after CXL. The results for MMP-1, -3, -7, -9, -13, and TIMP-2 were negative. CONCLUSION: CXL can lead to changes in the content of MMP-2 and TIMP-1 in the rabbit corneal stroma. The ratio of MMP-2/TIMP-1 remains higher versus baseline, indicating that MMP-2 is involved in the corneal pathophysiological process after CXL.