Enhanced expression of the cancer stem cell (CSC) marker, CD133, is closely associated with a higher rate of tumor formation and poor prognosis in hepatocellular carcinoma (HCC) patients. Despite its clinical significance, the molecular mechanism underlying the deregulation of CD133 during tumor progression remains to be clarified. Here, we report on a novel mechanism by which interleukin-6/signal transducer and activator of transcription 3 (IL-6/STAT3) signaling up-regulates expression of CD133 and promotes HCC progression. STAT3 activated by IL-6 rapidly bound to CD133 promoter and increased protein levels of CD133 in HCC cells. Reversely, in hypoxic conditions, RNA interference silencing of STAT3 resulted in decrease of CD133 levels, even in the presence of IL-6, with a concomitant decrease of hypoxia-inducible factor 1 alpha (HIF-1α) expression. Active STAT3 interacted with nuclear factor kappa B (NF-κB) p65 subunit to positively regulate the transcription of HIF-1α providing a mechanistic explanation on how those three oncogenes work together to increase the activity of CD133 in a hypoxic liver microenvironment. Activation of STAT3 and its consequent induction of HIF-1α and CD133 expression were not observed in Toll-like receptor 4/IL-6 double-knockout mice. Long-term silencing of CD133 by a lentiviral-based approach inhibited cancer cell-cycle progression and suppressed in vivo tumorigenicity by down-regulating expression of cytokinesis-related genes, such as TACC1, ACF7, and CKAP5. We also found that sorafenib and STAT3 inhibitor nifuroxazide inhibit HCC xenograft formation by blocking activation of STAT3 and expression of CD133 and HIF-1α proteins.