Although adenosine triphosphate-magnesium chloride (ATP-MgCl2) has demonstrated cytoprotective effects in a variety of adverse pathophysiologic conditions, its ability to alter radiation injury is unknown. The purpose of this study, therefore, was to assess the effects of ATP-MgCl2 on colorectal radiation injury after preoperative pelvic radiotherapy. Mixed-breed pigs (n = 36) received 4250 cGy preoperative external-beam pelvic radiotherapy (350 cGy fractions three times per week for 4 weeks). During radiotherapy, animals were randomly assigned to one of three treatment groups: (1) intravenous infusions of normal saline during radiotherapy, (2) intravenous ATP-MgCl2 (30 μmol/kg) during radiotherapy, or (3) intravenous ATP-MgCl2 (60 μmol/kg) during each radiotherapy session. After completion of radiotherapy and a 4-week rest period, animals underwent colorectal resection by either the two-layer hand-sewn (n = 18) or stapled end-to-end anastomosis technique (n = 18). Laser Doppler velocimetric readings were obtained to assess mural colonic blood flow after completion of anastomosis. A second laparotomy on postoperative day 5 or 11 was done to examine the following anastomotic parameters: (1) repeat laser Doppler velocimetry, (2) gross inflammatory scoring, (3) bursting pressure, (4) preoperative barium enema to identify leak or stenosis, (5) analysis of anastomotic hydroxyproline content, and (6) incidence of cutaneous injury in the radiation portals. ATP-MgCl2 administered intravenously at 60 μmol/kg led to (1) diminished colorectal seromuscular ischemia evidenced by laser Doppler velocimetric readings, (2) decreased skin and subcutaneous tissue injury in the treatment portals, (3) significantly decreased perianastomotic inflammatory reaction, and (4) increased early hydroxyproline content. There was no significant difference in the incidence of leakage or stenosis between the study groups, nor was the anastomotic bursting strength significantly different between the treatment groups. Therefore the administration of ATP-MgCl2 (60 μmol/kg) appears to offer significant cytoprotection from preoperative pelvic radiation therapy.