Chilling injury is one of the limiting steps for successful vitrification of cells, organs and tissue. Chilling injury is known to be caused by the decrease of temperature per se. Although some research has been done to study chilling injury in cryopreserved oocytes and sperm cells amongst others, the exact mechanism especially in the intact organ is yet unknown. Precision-cut liver slices (PCLS), contain all liver cell types and microstructures and closely resemble the organ from which they are derived. Therefore, it is an ideal model to study the mechanism of chilling injury in the intact organ. Using PCLS as a tool we aim at identifying the critical genes and signaling pathways responding to chilling injury by microarray analysis to enhance insight into its mechanism and to facilitate development of vitrification methods for integrated tissue and organs. Chilling injury was induced in rat PCLS loaded with cryoprotectant solutions (Sol Y or Sol Z, consisting of M22 without N-methyl-formamide and polyvinylpyrrolidone, and without or with sucrose respectively) and chilled to −15 °C for 10 min. At this temperature, no ice formation occurred as was shown by differential scanning calorimetry. The cryoprotectant solutions induced little or no damage as indicated by ATP levels. Principle Component Analysis indicated that gene expression changes induced in chilled PCLS compared to untreated controls were clearly distinguishable from those induced by the cryoprotectants alone. Pathway analysis in metacore revealed that immune innate and defense responses as well as cell cycle G1/S phase transition, MAP Kinase and lipid/fatty acid biosynthesis and metabolism were affected by chilling. Among the up-regulated pathways, cell surface receptor linked signaling pathways accounted for 68% of all processes, indicating that the cell membrane could be the primary site and sensor for chilling injury. Furthermore, we also found some indication that chilling might cause DNA damage, since DNA-damage inducible genes were up-regulated. In addition, chilling decreased gene expression of a set of enzymes required for cholesterol biosynthesis. Besides, pathway analysis indicated decreased fatty acid oxidation and unsaturated fatty acid biosynthesis. These observations could explain the decreased ratio of unsaturated fatty acid to saturated fatty acid and the decreased cholesterol content in cells after cryopreservation that was reported by others. Most importantly, we were able to observe the responses from the different cell types such as the increased differentiation of stellate cells, and immune responses (e.g. MHC class II activation) in Kupffer cells suggesting the involvement of all liver cell types in the injury. In conclusion, a broad spectrum of changes in the tissue at the gene level was identified that were specifically due to chilling, as well as previously unknown changes in the different cell types. This is the first report of a systematic investigation on the mechanism of chilling injury investigated in integrated tissue by microarray analysis under conditions in which other sources of injury are minimal.