BACKGROUND: Little is reported on human urine derived stem cells, and there is still no stable and efficient culture method until now. OBJECTIVE: To compare the effects of three kinds of culture media on the growth and proliferation of human urine derived stem cells, and to optimize the culture methods. METHODS: Human urine derived stem cells were isolated and purified using centrifugation method, and then cultured using adherent method in keratinocyte serum-free medium, progenitor cell culture medium, and urinary cell culture medium (equal-proportion mixture of keratinocyte serum-free medium and progenitor cell culture medium). Cell morphology was observed, and cell proliferation was detected by MTT method to draw a cell growth curve.RESULTS AND CONCLUSION: Human urine derived stem cells could be successfully cultured in these three kinds of culture media, which grew slowly in the keratinocyte serum-free medium, grew rapidly in the progenitor cell culture medium, and grew best in the urinary cell culture medium. To conclude, the urinary cell culture medium which can rapidly and efficiently culture target cells is the best choice for the growth of human urine derived stem cells.