Purpose: Many miRNAs have been identified as essential issues and core determining factors in tumor radiation.Recent reports have demonstrated that miRNAs and Toll-like receptors could exert reciprocal effects to control cancer development in various ways.However, a novel role of miR-15a/16 in enhancing radiation sensitivity by directly targeting TLR1 has not been reported, to our knowledge.Methods and Materials:Bioinformatic analyses, luciferase reporter assay, biochemical assays, and subcutaneous tumor establishment were used to characterize the signaling pathways of miRNA-15a/16 in response to radiation treatment.Results: First, an inverse correlation between the expression of miR-15a/16 and TLR1 protein was revealed in non-small cell lung cancer (NSCLC) and normal lung tissues.Next, we corroborated that miR-15a/16 specifically bound to TLR1 3UTR and inhibited the expression of TLR1 in H358 and A549 cells.Furthermore, miR-15a/16 down-regulated the activity of the NF-κB signaling pathway through TLR1.In addition,overexpression of miR-15a/16 inhibited survival capability and increased radiation-induced apoptosis, resulting in enhancement of radiosensitivity in H358 and A549 cells.Finally, subcutaneous tumor bearing NSCLC cells in a nude mice model was established, and the results showed that combined groups (miR-15a/16 + radiation) inhibited tumor growth more significantly than did radiation alone.Conclusions: We mainly elucidate that miRNA-15a/16 can enhance radiation sensitivity by regulating the TLR1/NF-κB signaling pathway and act as a potential therapeutic approach to overcome radioresistance for lung cancer treatment.