Around 12-15% of patients with locally advanced rectal cancer undergo a pathologically complete response (tumor regression grade 4) to long-course preoperative chemoradiotherapy; the remainder exhibit a spectrum of tumor regression (tumor regression grade 1-3). Understanding therapy-related transcriptional alterations may enable better prediction of response as measured by progression-free and overall survival, in addition to aiding the development of improved strategies based on the underlying biology of the disease. To this end, we performed high-throughput gene expression profiling in 40 pairs of formalin-fixed paraffin-embedded rectal cancer biopsies and matched resections following long-course preoperative chemoradiotherapy (discovery cohort). Differential gene expression analysis was performed contrasting tumor regression grades in resections. Enumeration of the tumor microenvironment cell population was undertaken using in silico analysis of the transcriptional data, and real-time PCR validation of NCR1 undertaken. Immunohistochemistry and survival analysis was used to measure CD56+ cell populations in an independent cohort (n=150). Gene expression traits observed following long-course preoperative chemoradiotherapy in the discovery cohort suggested an increased abundance of natural killer cells in tumors that displayed a clinical response to CRT in a tumor regression grade-dependent manner. CD56+ natural killer-cell populations were measured by immunohistochemistry and found to be significantly higher in tumor regression grade 3 patients compared with tumor regression grade 1-2 in the validation cohort. Furthermore, it was observed that patients positive for CD56 cells after therapy had a better overall survival (HR=0.282, 95% CI=0.109-0.729, χ(2)=7.854, P=0.005). In conclusion, we have identified a novel post-therapeutic natural killer-like transcription signature in patients responding to long-course preoperative chemoradiotherapy. Furthermore, patients with a higher abundance of CD56-positive natural killer cells post long-course preoperative chemoradiotherapy had better overall survival. Therefore, harnessing a natural killer-like response after therapy may improve outcomes for locally advanced rectal cancer patients. Finally, we hypothesize that future assessment of this natural killer-like response in on-treatment biopsy material may inform clinical decision-making for treatment duration.