The demand for organs to be transplanted increases pressure on procurement centers, to the detriment of organ quality, increasing complications. New preservation protocols are urgently needed, requiring an in-depth understanding of ischemia-reperfusion mechanisms. We performed a proteomic analysis using LC-MS/MS-TOF data analyzed through R software and Cytoscape’s ClueGO application, comparing the proteome of kidney endothelial cells, key cell type, subjected to 3, 6, 12, 19, and 24 h of cold ischemia and 6 h reperfusion. Critical pathways such as energy metabolism, cytoskeleton structure/transport system, and gene transcription/translation were modulated. Important time windows were revealed: a—during the first 3 h, central proteins were upregulated within these pathways; b—the majority of these upregulations were maintained until 12 h cold ischemia time (CIT); c—after that time, the overall decrease in protein expression was observed; d—at reperfusion, proteins expressed in response to cold ischemia were all downregulated. This shows that cold ischemia is not a simple slowing down of metabolism, as deep changes take place within the proteome on major pathways. Time-sensitive expression of key protein reveals possible quality biomarkers as well as potential targets for new strategies to maintain or optimize organ quality.