Abstract: Objective To evaluate the effect of different microstructures prepared by three-dimensional (3D) bioprinting on proliferation and viability of the murine epithelial stem cells in vitro. Methods 3D cell-laden microstructures were constructed using 3 different printing nozzles with diameters of 210, 340, and 420 μm. Fluorescence microscopy and the live/dead assay kit were used to observe the proliferation and viability of the murine epithelial stem cells in the microstructures. Results All the 3D cell-laden micro-structures were capable of promoting the proliferation of murine epithelial stem cells. In the 3 groups of micro-structures, the cell viability decreased significantly with time until 7 days after printing (P<0.01), but at 14 days after the printing, the cell viability increased significantly as compared with that at 7 days (P<0.01). The viability of the cells was significantly higher in the microstructure printed using a 420 μm nozzle than in the microstructures printed with 210 μm and 340 μm nozzles (P<0.01). Conclusion The microstructure printed with a 420 μm nozzle can stably promote the proliferation of murine epithelial stem cells and maintain a high level of cell viability, suggesting the feasibility of constructing tissue-engineered epidermis and full-thickness skin graft using 3D bioprinting technique.