Prohibitin 1 (PHB1) is a mitochondrial chaperone that regulates cell growth. Phb1 knockout mice exhibit liver injury and hepatocellular carcinoma (HCC). Phb1-knockout livers show induction of tumor growth-associated genes, H19 and insulin-like growth factor 2 (Igf2). These genes are controlled by the imprinting control region (ICR) containing CCCTC-binding transcription factor (CTCF)-binding sites. Since Phb1 knockout mice exhibited induction of H19 and Igf2, we hypothesized that PHB1-mediated regulation of the H19-Igf2 axis might control cell proliferation in normal hepatocytes. H19 and Igf2 were induced (8-20-fold) in 3-week old Phb1-knockout livers, in Phb1 siRNA-treated AML12 hepatocytes (2-fold) and HCC cell lines compared to control. Phb1 knockdown lowered CTCF protein in AML12 by ~30% compared to control. CTCF over-expression lowered basal H19 and Igf2 expression by 30% and suppressed Phb1 knockdown-mediated induction of these genes. CTCF and PHB1 co-immunoprecipitated and co-localized on the ICR element and Phb1 knockdown lowered CTCF ICR-binding activity. The results suggest that PHB1 and CTCF cooperation may control the H19-Igf2 axis. Human HCC tissues with high levels of H19 and IGF2 exhibited a 40-50% reduction in PHB1 and CTCF expression and their ICR binding activity. Silencing Phb1 or over-expressing H19 in the mouse HCC cell line, SAMe-D, induced cell growth. Blocking H19 induction prevented Phb1 knockdown-mediated growth; whereas H19 over-expression had the reverse effect. Interestingly H19 silencing induced PHB1 expression. Taken together, our results demonstrate that the H19-Igf2 axis is negatively regulated by CTCF-PHB1 co-operation and H19 is involved in modulating PHB1s growth suppressive effect in the liver.