Abstract Hepatocellular carcinoma (HCC) is the second most lethal cancer worldwide. Frequent relapse and drug resistance accounts for the poor prognosis, which could be attributed by the presence of cancer stem cells (CSCs), a subpopulation that harbor the properties of self-renewal, clonal tumor initiation and long-term repopulation capacity. Toll like receptors (TLRs) are widely expressed on immune cells and TLR/Interleukin-1 receptor associated kinase (IRAK) pathway was originally best known for their role in the induction of innate immune responses, but it was recently found to be frequently up-regulated in various cancers, although its role in cancer development remains largely unexplored. By RNA sequencing analysis of 16 pairs of HCC clinical samples, we found IRAK1 to be significantly overexpressed in HCC among all members of IRAK family. By quantitative PCR and western blot analyses, overexpression of IRAK1 at both mRNA and protein level was confirmed, which correlated with advanced tumor stage (p = 0.04). By lentiviral-based over-expression and knockdown approaches, we demonstrated that IRAK1 promoted cell proliferation and invasiveness. Interestingly, IRAK1 regulated CSC properties including tumorigenicity, self-renewal, drug resistance and expression of liver CSC markers including CD24 and CD47. To further examine the therapeutic potentials of targeting IRAK1 in HCC, we treated HCC cells with IRAK1/4 inhibitor. Suppression of IRAK1 at 10μM not only consistently inhibited CSC properties, but also augmented the effect of sorafenib, a molecularly targeted drug for treatment of advanced HCC. The cancer-promoting and drug desensitization effect of IRAK1 could be partly explained by activation of NF-κB signaling pathway as well as down-regulation of apoptotic cascade. Conclusion: Overexpression of IRAK1 in HCC promotes cancer progression by enhancing proliferation and invasiveness; Importantly, IRAK1 may promote cancer stemness and confers resistance to sorafenib. All in all, targeting IRAK1 with specific small molecule inhibitor alone or in combination with other drugs may be a novel therapeutic regimen for treatment of HCC. Citation Format: Yik Ling Bowie Cheng, Oi Lin Irene Ng, Kin Wah Terence Lee. Inhibition of TLR/IRAK pathway in hepatocellular carcinoma augmented therapeutic responses. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5152.