Increased activity of signal transducer and activator of transcription 3 (STAT3) is common in human malignancies, including colorectal cancers (CRC). We have recently reported that STAT3 gene expression correlates with resistance of CRC cell lines to 5-fluorouracil (5-FU) based chemoradiotherapy. This is of considerable clinical importance, because a large proportion of rectal cancers are resistant to preoperative multimodal treatment. To test whether STAT3 contributes to chemoradiotherapy-resistance, we first confirmed that STAT3 protein expression correlated positively with increasing resistance. While STAT3 was not constitutively active, stimulation with Interleukin-6 (IL-6) resulted in remarkably higher expression levels of phosphorylated STAT3 in chemoradiotherapy-resistant cell lines. A similar result was observed when we determined IL-6-induced expression levels of phosphorylated STAT3 following irradiation. Next, STAT3 was inhibited in SW480 and SW837 using siRNA, shRNA, and the small-molecule inhibitor STATTIC. Successful silencing and inhibition of phosphorylation was confirmed using Western blot analysis and a luciferase reporter assay. RNAi-mediated silencing as well as STATTIC treatment resulted in significantly decreased clonogenic survival following exposure to 3 µM of 5-FU and irradiation in a dose-dependent manner, with dose-modifying factors of 1.3 to 2.5 at a surviving fraction of 0.37. Finally, STAT3 inhibition led to a profound chemoradiotherapy-sensitization in a subcutaneous xenograft model, with a significantly delayed tumor re-growth in STATTIC-treated mice compared with control animals. These results highlight a potential role of STAT3 in mediating treatment resistance, and provide first proof of concept that STAT3 represents a promising novel molecular target for sensitizing resistant rectal cancers to chemoradiotherapy. © 2013 Wiley Periodicals, Inc.