Frequent relapse and drug resistance in patients with hepatocellular carcinoma (HCC) can be attributed to the existence of tumor-initiating cells (TIC) within the tumor bulk. Therefore, targeting liver TICs may improve the prognosis of these patients. From transcriptome sequencing of 16 pairs of clinical HCC samples, we report that interleukin-1 receptor-associated kinase 1 (IRAK1) in the TLR/IRAK pathway is significantly upregulated in HCC. IRAK1 overexpression in HCC was further confirmed at the mRNA and protein levels and correlated with advanced tumor stages and poor patient survival. Interestingly, IRAK4, an upstream regulator of IRAK1, was also consistently upregulated. IRAK1 regulated liver TIC properties, including self-renewal, tumorigenicity, and liver TIC marker expression. IRAK1 inhibition sensitized HCC cells to doxorubicin and sorafenib treatment in vitro via suppression of the apoptotic cascade. Pharmacological inhibition of IRAK1 with a specific IRAK1/4 kinase inhibitor consistently suppressed liver TIC populations. We identified aldo-keto reductase family 1 member 10 (AKR1B10) as a novel downstream target of IRAK1, which was found to be overexpressed in HCC and significantly correlated with IRAK1 expression. Knockdown of AKR1B10 negated IRAK1-induced TIC functions via modulation of the AP-1 complex. Inhibition of IRAK1/4 inhibitor in combination with sorafenib synergistically suppressed tumor growth in an HCC xenograft model. In conclusion, targeting the IRAK4/IRAK1/AP-1/AKR1B10 signaling pathway may be a potential therapeutic strategy against HCC. Significance: IRAK4/IRAK1/AP-1/AKR1B10 signaling pathway regulates cancer stemness and drug resistance and may be a novel therapeutic target in HCC.