To elucidate the mechanism whereby distigmine, an underactive bladder remedy, potentiates urinary bladder contractions long-lastingly, the inhibition of recombinant human acetylcholinesterase (rhAChE) by distigmine was investigated. A centrifugal ultrafiltration device, Nanosep^® 10K, was used to separate rhAChE and a bound inhibitor from an unbound inhibitor, reaction substrate, and reaction product. This allowed the same aliquot of rhAChE to be repeatedly assayed for up to 48 h to confirm the long-lasting binding of an inhibitor. Cholinesterase (ChE) inhibitors, distigmine, pyridostigmine, neostigmine, and ambenonium, were tested. The dissociation rate constant ( k _diss) and dissociation half-life ( t _1/2) of each inhibitor were determined based on the changes in rhAChE activity. Within 2–4 h after removing pyridostigmine, neostigmine, or ambenonium, the rhAChE activity was restored to the control levels. The k _diss values for pyridostigmine, neostigmine, and ambenonium were calculated to be 0.51±0.05, 0.66±0.03, and 1.41±0.08 h⁻¹, and the t _1/2 values were calculated to be 1.36, 1.05, and 0.49 h, respectively. With distigmine, the rhAChE activity initially dropped to 17% of that in the control and then slowly recovered to only 50% by 48 h after drug removal. The k _diss and t _1/2 values of distigmine were calculated to be 0.012±0.001 h⁻¹ and 57.8 h, respectively. Based on the t _1/2 values, distigmine was judged to dissociate from acetylcholinesterase (AChE) 40–120-fold slower than the other ChE inhibitors did. This may explain the long-lasting potentiation of urinary bladder contractions and motility by distigmine as a treatment for an underactive bladder.