【Abstract】Objective To investigate the effects of threedimensional (3D) rat tail collagen I culture on the drug resistance of human breast cancer cell MCF7, including the drug sensitivity, identification of stem cell surface markers, cell cycle, mRNA expressions of stem cell related genes and drug resistance related ABC transporters. Methods Two experimental groups were established as 2D group and 3D rat tail collagen I group according to the culture methods. Cytotoxicity of antitumor drugs of Paclitaxel and Cisplatin in 2D and 3D cultures of MCF7 cells was measured by CCK8 assay. CD44+/CD24subtype cells and cell cycle were analyzed by flow cytometry. qPCR was used to determine the expressions of stem cell related genes SOX2 and KLF4, and drug resistance related ABC transporters ABCC1 and ABCB1. ResultsCompared to 2D group, the drug resistance of MCF7 cells in 3D group enhanced significantly. Flow cytometric analysis results indicated that the percentage of CD44+/CD24subtype cells was higher than that of 2D group and cell cycle showed G1 phase arrest. The results of qPCR revealed that the expression levels of stem cell related genes SOX2 and KLF4, and the drug resistance related ABC transporters ABCC1 and ABCB1 were significantly unregulated. Conclusion MCF7 cells cultured in 3D rat tail collagen I showed an increase of drug resistance accompanied by the upregulation of cell stemness and G1 phase arrest. Rat tail collagen I could be used as a new promising scaffold material for studies of drug screening and drug resistance of cancer cells.