BACKGROUND:: Two-dimensional (2D) culture model is a common tool for tumor research and drug screening in vitro; however, there are some limitations. OBJECTIVE:: To establish a three-dimensional (3D) culture model of cholangiocarcinoma cells and to compare the difference in cell growth and metabolism between 2D and 3D culture systems. METHODS:: Cholangiocarcinoma cell line QBC939 was cultured in 2D and Cytodex-3 or Cytopore-2 microcarrier-based 3D culture systems. Cell morphology was observed under inverted light microscopy. Total cell number, glucose and lactate concentrations were determined, and the kinetic constants of cell growth and metabolism were calculated. RESULTS AND CONCLUSION:: In comparison with 2D and Cytodex-3-based 3D culture systems, Cytopore-2-based 3D culture system achieved higher cell adhesion rate, higher cell density, and lower specific glucose consumption and specific lactate production rates (P < 0.05). In addition, cells were uniformly distributed in Cytopore-2 microcarriers. Therefore, Cytodex-3 or Cytopore-2 microcarriers can be used to establish cholangiocarcinoma 3D culture model, and Cytopore-2 3D culture system is better than Cytodex-3 system.