Cold preservation tissue injury remains an unsolved problem during small intestinal transplantation. Pituitary adenylate cyclase-activating polypeptide (PACAP) plays a central role in the intestinal physiology. The aim of our study was to compare the cold ischemic injury in wild-type and PACAP-38 deficient mice after small bowel cold storage. Cold ischemia was produced with small bowel preservation in a University of Wisconsin solution at 4°C in wild-type (n = 35) mice for 1 h (GI), for 3 h (GII), and for 6 h (GIII); and in PACAP-38 deficient (n = 35) mice for 1 h (GIV), for 3 h (GV), and for 6 h (GVI). Small bowel biopsies were collected after laparotomy (Control) and at the end of the ischemia periods. To determine oxidative stress parameters, malondialdehyde (MDA), reduced glutathione (GSH), and superoxide dismutase (SOD) were measured. Tissue damage was analyzed by qualitative and quantitative methods on hematoxylin/eosin-stained sections. In PACAP-38 deficient animals, tissue lipid peroxidation was elevated. These changes were significant after 6 h (153.04 ± 7.2) compared to sham-operated (110.44 ± 5.5) and compared to wild-type results (120.0 ± 1.1 µmol/g, p < 0.05). Meanwhile, the capacity and activity of the endogenous antioxidant system decreased significantly after 3 and 6 h preservation (GSH: 808.7 ± 5.2; 720.4 ± 8.7 vs. 910.4 ± µmol/g; SOD: 125.1 ± 1.4; 103.3 ± 1.9 vs. 212.11 ± 5.8 IU/g). Qualitative and quantitative histological results showed destruction of the mucous, submucous layers, and crypts in PACAP-38 deficient mice compared to wild-type tissues. These processes depended on the time of the cold preservation periods. Our present study showed that the presence of PACAP-38 in the small bowel tissue has a key role in the protection against intestinal cold preservation injury.