Updated project metadata. Intestinal ischemia-reperfusion injury (IR) is a severe clinical condition, and unraveling its pathophysiology is crucial in order to improve therapeutic strategies and reduce the high morbidity and mortality rates. Here, we studied the dynamic proteome and phosphoproteome in human intestine during ischemia and reperfusion, using LC-MS analysis to gain quantitative information of thousands of proteins and phosphosites, as well as MS imaging to obtain spatial information. We identified a significant decrease in abundance of proteins related to intestinal absorption, microvillus and cell junction, whereas proteins involved in innate immunity, in particular the complement cascade, and extracellular matrix organization increased in abundance after IR. Differentially phosphorylated proteins were involved in RNA splicing events and cytoskeletal and cell junction organization. In addition, our analysis points to MAPK and CDK families to be active kinases during IR. Finally, MALDI-TOF MS imaging presented peptide alterations in abundance and distribution, which resulted, in combination with FTICR-MS imaging and LC-MS, in the identification of additional proteins related to RNA splicing, the complement cascade and extracellular matrix organization. This study expanded our understanding of the molecular changes that occur during IR in human intestine, and highlights the value of the complementary use of different MS-based methodologies.