Human aldo-keto reductase 1C3, type 2 3α-hydroxysteroid dehydrogenase (HSD)/type 5 17β-HSD (AKR1C3) is known to be involved in steroid, prostaglandin and lipid aldehyde metabolism. The role of AKR1C3 in the radiosensitivity to X-rays of human non-small-cell lung cancer (NSCLC) cells was explored. In this study, a specific small interfering RNA (siRNA) to target the AKR1C3 gene was used. A suite of readouts including cell survival were determined using a colony formation assay; apoptosis evaluated by Annexin V expression levels, irradiation-induced cytotoxicity established using a MTT cell viability assay and cell cycle distribution measured by flow cytometry were used in characterizing the role of the AKR1C3 gene. Although AKR1C3 was significantly overexpressed in both our radioresistant subclone cells and NSCLC tissues, a specific AKR1C3 siRNA significantly enhanced cell radiosensitivity and was concomitant with decreased expression of this gene. Furthermore, reduced interleukin-6 (IL-6)-mediated radioresistance was observed when siRNA was used to knock down AKR1C3 activity. This AKR1C3-mediated radioresistance was correlated with an arrest in the G2/M cell cycle and a decreased induction of apoptosis. AKR1C3 may present a potential therapeutic target in addressing radioresistance of NSCLC, and in particular in IL-6-mediated radioresistance.