This study was designed to investigate the effects of α-defensin 1 on electrical field stimulation (EFS)-induced contractions in isolated rat bladder detrusor muscles. We evaluated the effects of α-defensin 1 (50 pM˜5 nM) on EFS-induced contractions in the detrusor smooth muscles from 35 rats (2–30 Hz). Bladder strips were pretreated with α-defensin 1 and then changes of contractions by adenosine 5′-triphosphate (ATP) were observed. Moreover, after pretreatment with α-defensin 1 for 10 min, changes in concentration-response curves to hydrogen peroxide (H2O2) were investigated. Alpha-defensin 1 has increased EFS-induced contractions, significantly, and the contractile response to ATP (1,2,5,10 mM) was also increased significantly when strips were pretreated with α-defensin 1. In addition, alpha-defensin 1 increased H2O2-induced contractions. The present study demonstrates that α-defensin 1 increases EFS-induced contractions of rat detrusor muscles via purinergic contraction coupled with the Rho kinase pathway.