The protein 14-3-3ζ contributes important regulatory functions in several cellular processes via binding to phosphorylated serine/threonine residues, which promotes cell cycle progression, cell proliferation and anti-apoptosis in multiple types of cancer. The aim of the present study was to investigate the functions of 14-3-3ζ in cholangiocarcinoma (CCA) progression and elucidate the molecular mechanism of 14-3-3ζ expression-mediated protein kinase B (Akt) phosphorylation and chemosensitivity in CCA cells. In the present study, 14-3-3ζ expression was investigated in clinical specimens using immunohistochemistry and compared with the clinicopathological features of patients with CCA. The association between 14-3-3ζ and phosphorylated Akt (pAkt) was determined among the tissues of the same patients using bivariate correlation analysis. The effects of 14-3-3ζ suppression on CCA cell function and gemcitabine sensitivity were investigated using small interfering RNA (siRNA). It was identified that 14-3-3ζ expression was positively correlated with pAkt (P=0.013) and that increased expression of 14-3-3ζ and pAkt were significantly associated with poor overall survival rate and metastasis (P=0.025 and 0.006, respectively). Downregulation of 14-3-3ζ using siRNA in CCA cell lines decreased cell proliferation, resulting in the inhibition of pAkt activity and increasing the protein level of the cell cycle inhibitor p27. The suppression of 14-3-3ζ enhanced the inhibitory effect of gemcitabine on CCA cell proliferation by inducing apoptotic cell death. Taken together, the results of the present study indicated that 14-3-3ζ is a potential target for CCA and may serve as a novel therapeutic approach to enhance chemosensitivity in the treatment of CCA.