We investigated the current characteristics of large conductance voltage and Ca2+-activated K+(BK) channels in human urine-derived stem cells (hUSCs) and the effect of BK channels on proliferation and differentiation of hUSCs. Fresh human urine (n= 6) was collected from healthy donors to isolate hUSCs. Human KCNMA1 gene silencing U6 shRNA was used to down regulate the expression of BK in hUSCs. IBTX (BK channel antagonist) and NS1619 (BK channel agonist) were used to examine the effect of BK channels on hUSCs. Whole cell patch-clamping was employed to detect the current of BK channels. Flow cytometry, immunofluorescence, and western blotting were used to analyze the cell cycle and related protein levels. The results showed that the activities of BK channels were significantly decreased in P5, P7 and induced hUSCs (endothelial, urothelial and smooth muscle cells) compared with P3 hUSCs when normalized to the cell capacitance. In addition, the average BK channel current density of hUSCs was significantly decreased upon silencing BK channel expression by hnRNA. Apoptosis rates of hUSCs in iberiotoxin (IBTX) and hnRNA treatment groups were significantly increased compared with the control group, whereas treatment with BK agonist NS1619 decreased apoptosis rates. Compared with the control group, hUSCs in S phase were significantly decreased in IBTX and hnRNA treatment groups. In conclusion, BK channels play an important role in maintaining the proliferation and differentiation of hUSCs. Overexpression of BK channels in hUSCs be provide a basis for future clinical application to an overactive bladder.