Family with sequence similarity 189, also known as COTE1, has been found to be significantly upregulated in hepatocellular carcinoma (HCC) specimens and cell lines and is associated with tumor size and differentiation. Furthermore, COTE1 contributes to hepatocellular carcinogenesis. The overexpression of COTE1 enhanced in vitro cell viability and colony formation in soft agar, and in vivo tumorigenicity of HCC-derived Focus and Huh7 cells. In contrast, COTE1 knockdown via RNAi markedly suppressed these phenotypes in YY-8103 and WRL-68 HCC cell lines. Mechanistic analyses indicated that COTE1 physically associated with WW domain-containing oxidoreductase (WWOX) and modulated WWOX tyrosine phosphorylation. The ectopic overexpression of COTE1 inhibited the WWOX-p53 signaling pathway by reducing the phosphorylation of WWOX at the Tyr33 residue in Focus cells. Conversely, COTE1 silencing activated tyrosine 33 phosphorylation of WWOX and induced WWOX-p53 mediated mitochondrial apoptosis in WRL-68 cells. In addition, COTE1 upregulation in Huh7 cells blocked the WWOX-cyclin D1 pathway via dephosphorylation of WWOX Tyr287, stimulating cell cycle progression whereas phosphorylation of Tyr287 of WWOX induced by COTE1 silencing resulted in activation of WWOX-cyclin D1 signaling, leading to cell cycle arrest in YY-8103 cells. Together, our findings suggest that the cytoplasmic protein COTE1 contributes to HCC tumorigenesis by regulating cell proliferation through the modulation of WWOX signaling.