Distigmine bromide (distigmine) is a carbamate cholinesterase (ChE) inhibitor, which is mainly used for the treatment of myasthenia gravis. Distigmine is also used in Japan for the treatment for underactive bladder and glaucoma. The effectiveness of distigmine for underactive bladder treatment has been confirmed by many clinical reports, and this effect is thought to be caused by potentiating urinary bladder smooth muscle contraction due to inhibition of acetylcholine degradation during micturition. However, the pharmacological effects of distigmine on urinary bladder smooth muscle have not been well studied. The most distinctive pharmacological feature of distigmine is that it shows long-lasting effects than other ChE inhibitors; however, few studies have investigated the persistence of the enhancing effect of distigmine on the contractile function of urinary bladder smooth muscle. Moreover, this mechanism remains unclear. In this review, we present our findings on the mechanism of the potentiating effect of distigmine on isolated guinea pig urinary bladder smooth muscle contraction. We also discuss the long-lasting potentiating effect of distigmine on urinary bladder motility and the mechanism of these effects using guinea pig urinary bladder smooth muscle in vivo and in vitro. In addition, we present our investigations on the long-lasting mechanism of distigmine using recombinant human acetylcholinesterase.