The Na+/Ca2+ exchangers (NCXs) are expressed in a variety of cell types and participate in the regulation of cytosolic Ca2+ mobilization. In mammals, three NCX isoforms (NCX1, NCX2, and NCX3) have been identified as products of the SLC8 gene family. Among alternative splicing variants of NCX1, NCX1.3 and NCX1.7 are the predominant isoforms expressed in smooth muscle tissues. NCX is thought to be a key molecule in the regulation of cytosolic Ca2+ dynamics in smooth muscles. The relative importance of the two Ca2+ transport modes of NCX activity leading to Ca2+ efflux (forward mode) and Ca2+ influx (reverse mode) in smooth muscle, however, remain unclear. Unexpectedly, spontaneous contractions of urinary bladder smooth muscle were enhanced in transgenic mice overexpressing NCX1.3 (NCX1.3tg/tg mice) compared to wild-type (WT) mice. For this reason, the present study was undertaken to elucidate the contribution of Ca2+ influx via reverse mode NCX activity to the control of membrane electrical excitability, cytosolic Ca2+ mobilization in urinary bladder myocytes, urinary bladder smooth muscle contractility, and urinary bladder function. All experiments were approved by the Ethics Committee of the Nagoya City University and were conducted in accordance with the Guide for the Care and Use of Laboratory Animals of the Japanese Pharmacological Society. The enhanced spontaneous contractions of urinary bladder smooth muscle from NCX1.3tg/tg mice were attenuated by KB-R7943 or SN-6. Whole-cell outward NCX current sensitive to KB-R7943 or Ni2+ was readily detected in urinary bladder smooth muscle cells from NCX1.3tg/tg mice, but not in WT mice. The half duration of spontaneous transient outward currents (STOCs) in myocytes of NCX1.3tg/tg mice were significantly larger than those of WT mice. Spontaneous Ca2+ transients (Ca2+ sparks) in myocytes of NCX1.3tg/tg mice were larger and frequently resulted in propagating events and global elevations in cytosolic Ca2+ concentration (Ca2+ waves). 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 NCX1.3tg/tg (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 contraction in urinary bladder smooth muscles. These findings suggest the possibility that NCX is a candidate molecular target for overactive bladder (OAB) therapy.