Cornea transplantation is a widely performed procedure globally. However, storing donor corneas for extended periods in Optisol-GS storage medium at 4 ℃ can lead to reduced endothelial cell viability, causing severe stromal edema and opacification. This negatively impacts transplantation success. The corneal stroma, crucial for maintaining shape and transparency, was subjected to proteomic analysis after 7- or 14-day preservation. Among 1,545 identified proteins, 1,113 were quantifiable, with 255 significantly regulated in longer preservation. Proteins related to ocular surface immunomodulation decreased, while those involved in extracellular matrix reorganization and fibrosis increased after 14 days. The observed upregulation of extracellular matrix structural proteins and growth factor signaling suggests the occurrence of stromal fibrosis, potentially leading to compromised tissue clarity and vision impairments. This study provides novel insights into the impact of preservation duration on the corneal stroma from a proteomic perspective. Our findings may aid future research in developing long-term preservation techniques and enhancing the quality of preserved corneas, thereby maximizing their clinical usefulness.