Background: Mechanisms of radioresistance in rectal cancer remain unclear. Objectives: To determine mechanisms of radioresistance in rectal cancer cells and to assess the role of the nitric oxide donor DETANONOate as a radiosensitizing agent. Methods: Survival was determined by clonogenic assays, apoptosis by PARP-1cleavage, and phenotypic differences byWestern blot analysis.SCID mice bearing HT-29 xenografts were treated with ionizing radiation (IR) [2.0 Gy x 5], DETANONOate [0.4 mg/kg i.p.], or combination treatment. Results: Colorectal cancer HT-29-p53-null cells were resistant and HCT-116-p53 wild-type cells sensitive to IR, which correlated with cleaved PARP-1. Increased levels of p21 occurred in HCT-116 cells, while Bcl-2 and survivin were elevated inHT-29 cells. Radiosensitization was achieved with a substantial elevation of cleaved PARP-1 in DETANONOate-HT-29-treated versus control cells, which was accompanied by elevation of p21, p27, and BAX, and a concomitant decrease in Bcl-2. SCID mice bearing HT-29 xenografts demonstrated a 37.6%, 51.1%, and 70.1% inhibition in tumor growth in mice receiving IR, DETANONOate, and combination treatment versus control, respectively. Conclusions: RadioresistantHT-29 cells are p53-null and have substantially decreased levels of p21.DETANONOate radiosensitized HT-29 cells in vitro and in vivo by an additive effect in apoptosis.