Baicalein, the primary flavonoid derived from the roots of Scutellaria baicalensis Georgi, is known to exhibit anticancer effects against different types of cancer. However, the anticancer activity and underlying mechanisms of baicalein on cholangiocarcinoma (CCA) have not been elucidated. Here, our results showed that baicalein inhibited the viability and proliferation of CCA cells in a dose and time-dependent manner. Besides, baicalein also induced cell apoptosis and cell cycle arrest by regulating cell cycle associated proteins and Bcl-2 family proteins expression. More importantly, baicalein also suppressed the migration and invasion by reducing the expression of MMP-2 and MMP-9 proteins, as well as decreased the aerobic glycolysis of CCA cells by reducing Hexokinase and Glucose-6-phosphate. In the mechanistic study, the phosphorylation of I kappa B alpha, AKT and STAT3 were all significantly decreased by baicalein in vitro and in vivo. Furthermore, baicalein also inhibited the tumor growth in subcutaneous xenograft model of mice. These findings indicate that baicalein has potent anticancer activities by suppressing multiple malignant phenotypes and most probably through inhibiting the activation of the AKT/NF-kappa B and STAT3 signaling pathway. Thus, it has the potential to be developed as an anticancer agent to enhance clinical standards of care for patients with CCA.