Three-dimensional (3D) cell culture may provide the architectures similar to the in vivo natural extracellular matrix condition for in vitro cultured cells. In this work, a 3D collagen scaffold was used to culture the adenoid cystic carcinoma (ACC) cells. The results showed that the 3D scaffold not only induced the diversification of cell morphologies but also increased the cell proliferation. The transcription of matrix metalloproteinase and epithelial–mesenchymal transition were significantly increased in the cells cultured in 3D collagen scaffolds. In addition, the expression of cancer stem cell (CSC) markers of Sox2 and Oct4 were higher than that in 2D cultured cells. The 3D cultured ACC-83 cells showed more resistance to chemotherapeutic drugs. Thus, the 3D collagen scaffold could provide a useful model for CSCs study and anticancer therapeutics research in vitro. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 102B: 772–780, 2014.