The 14-3-3sigma gene has been implicated in G2/M cell cycle arrest by p53, and the loss of 14-3-3sigma protein expression has been reported in diverse human cancers. However, the role of 14-3-3sigma in the signaling pathway of the cell cycle in the progression of intrahepatic cholangiocarcinoma has not been well understood. To clarify the role of 14-3-3sigma, we examined the protein expressions of 14-3-3sigma, cyclin B1, and p53 in 93 cases of intrahepatic cholangiocarcinoma by immunohistochemical staining. We also examined the correlation between these expressions and survival rate and clinicopathologic factors such as sex, age, tumor grade (ie, pathologic differentiation, tumor size, lymphatic permeation, vascular invasion, perineural invasion, lymph node metastasis), and tumor stage. Positive 14-3-3sigma protein expression (>30% of tumor cells) was observed in 67.7% (63/93) of cases of intrahepatic cholangiocarcinoma and was inversely correlated with cyclin B1 expression. No correlation was found between 14-3-3sigma expression and p53 expression or clinicopathologic factors; however, decreased 14-3-3sigma expression was an independent prognostic factor by multivariate survival analysis (P = .0282). Extensive methylation of 14-3-3sigma was found by methylation-specific polymerase chain reaction and sequence; however, no significant correlation was detected between methylation states and protein expression. These results indicate that depressed 14-3-3sigma protein is involved in the uncontrolled cell cycle in intrahepatic cholangiocarcinoma and that the decreased expression of 14-3-3sigma protein is a significant indicator of poor prognosis for patients with intrahepatic cholangiocarcinoma.