Background: The response to neoadjuvant chemoradiation in rectal cancer is variable and unpredictable. Resistance to chemoradiation has been directly correlated with the levels of the inhibitors of apoptosis (IAPs) in several malignancies. Because smac-DIABLO is a pro-apoptotic gene product that directly inhibits the activity of the IAPs, molecules with similar activity might radiosensitize rectal tumors with phenotypes that express high levels of IAPs. This study was undertaken to assess the radiosensitizing properties of the smac mimetic JP-1201 in radioresistant HT-29 colorectal cancer cells in vitro and established xenografts in SCID mice. Methods: Survival was determined by clonogenic assays. PARP-1, caspase-8 cleavage, and IAP levels were assessed by Western blot analysis. SCID mice bearing HT-29 xenografts were treated with ionizing radiation: 2.0 Gy × 5; (n = 6), JP-1201 (5.0 mg/Kg i.p., n = 5) or combination treatment (n = 7) and compared to control (n = 8). DNA repair mechanisms were interrogated by γH2AX positive foci. Results: Pretreatment of HT-29 cells with JP-1201 (5.0 μM) prior to ionizing radiation (IR) significantly decreased the survival of these cells. SCID mice bearing HT-29 xenografts demonstrated no difference in tumor load in the group receiving exclusively JP-1201 versus control. At the end of the treatment (day 40), a 46% reduction of tumor load was observed in the IR+JP-1201-treated group compared to the IR-only treated group. Radiosensitization was achieved with a substantial elevation of cleaved PARP-1 in JP-1201- treated HT-29 cells versus control cells with a concomitant decrease of XIAP, but not of survivin or cIAP1/2. JP-1201-treated HT-29 cells had a reduced ability to repair double-stranded DNA breaks (DSBs). Conclusion: The smac mimetic JP-1201 decreased the survival of HT-29 cells and tumor growth by an additive effect in apoptosis and a reduction in the level of XIAP and an impairment of DNA repair mechanisms. The pathways leading to this response need to be further investigated.