Small bowel is one of the most sensitive organs to ischemia-reperfusion injury, which is a significant problem during transplantation. Pituitary adenylate cyclase-activating polypeptide (PACAP) has cytoprotective effect in ischemic injuries of various tissues. The aim of our study was to measure changes of PACAP-38 and PACAP-27 immunoreactivities and cytokine levels in intestinal grafts stored in PACAP-38-containing preservation solution. Small bowel autotransplantation was performed on male Wistar rats. Grafts were stored in University of Wisconsin (UW) solution at 4 °C for 1 h (group (G)I), for 3 h (GII), and for 6 h (GIII) and in PACAP-38-containing UW solution for 1 h (GIV), for 3 h (GV), and for 6 h (GVI). After preservation, performing vessel anastomosis reperfusion began, which lasted 3 h in each group. Tissue biopsies were collected after laparotomy (control) and at the end of the reperfusion periods. Intestinal PACAP-38 and PACAP-27 immunoreactivities were measured by radioimmunoassay. To measure cytokines from tissue homogenates, we used rat cytokine array and Luminex Multiplex Immunoassay. Levels of PACAP-38 and PACAP-27 immunoreactivity decreased after 1 and 3 h preservation compared to control levels. This decrease was significant following 6 h cold storage (p < 0.05). Values remained significantly higher in grafts stored in PACAP-38-containing UW. Cytokine array revealed that expression of the soluble intercellular adhesion molecule-1 (CD54) and L-selectin (CD62L/LECAM-1) was increased in GIII. Both 6 h cold storage in PACAP-38-containing UW solution and 3 h reperfusion caused strong reduction in these cytokines activation in GVI. RANTES (CCL5) levels were increased in all groups. Strong activation of the tissue inhibitor of metalloproteinase-1 was in GIII. However, PACAP-38-containing cold storage could decrease its activation in GVI. Furthermore, strong activation of the tissue inhibitor of metalloproteinase-1 was detected in 6 h preserved grafts without PACAP-38 (GIII). PACAP-38-containing cold storage could decrease its activation in GVI. Our present study showed that PACAP-38 and PACAP-27 immunoreactivities decreased in a time-dependent manner during intestinal cold preservation, which could be ameliorated by administration of exogenous PACAP-38 to the preservation solution. Moreover, PACAP-38 could attenuate tissue cold ischemic injury-induced changes in cytokine expression.