The Na+/Ca2+ exchanger (NCX) is thought to be a key molecule in the regulation of cytosolic Ca2+ dynamics. The relative importance of the two Ca2+ transport modes of NCX activity leading to Ca2+ efflux (forward) and influx (reverse) in smooth muscle, however, remains unclear. Unexpectedly, spontaneous contractions of urinary bladder smooth muscle (UBSM) were enhanced in transgenic mice overexpressing NCX1.3 (NCX1.3tg/tg). The enhanced activity was attenuated by KB-R7943 or SN-6. Whole cell outward NCX current sensitive to KB-R7943 or Ni2+ was readily detected in UBSM cells from NCX1.3tg/tg but not wild-type mice. Spontaneous Ca2+ transients in myocytes of NCX1.3tg/tg were larger and frequently resulted in propagating events and global elevations in cytosolic Ca2+ concentration. Significantly, NCX1.3tg/tg mice exhibited a pattern of more frequent urination of smaller volumes and this phenotype was reversed by oral administration of KB-R7943. On the other hand, KB-R7943 did not improve it in KB-R7943-insensitive (G833C-)NCX1.3tg/tg mice. We conclude that NCX1.3 overexpression is associated with abnormal urination owing to enhanced Ca2+ influx via reverse mode NCX leading to prolonged, propagating spontaneous Ca2+ release events and a potentiation of spontaneous UBSM contraction. These findings suggest the possibility that NCX is a candidate molecular target for overactive bladder therapy.