A new muscarinic receptor antagonist, 5-hydroxymethyl tolterodine (5-HMT), was successfully encapsulated into PLGA microspheres. With an increase of PLGA concentration from 15% to 40%, encapsulation efficiency of 5-HMT increased from 55.39% to 76.32%, and the particle size of microsphere increased from 34.33 to 70.48 µm. Increasing the homogenisation speed from 850 to 2300 rpm, the particle size was reduced about 65%.The in vitro and in vivo studies in beagle dogs show that the release profile of 5-HMT-loaded microspheres (5-HMT MS) prepared with 503H is characterised by a low initial burst followed by slow release that lasted for 2 weeks. A C(max) of 1.617 ± 0.392 ng/mL was found on the sixth day. When evaluated for inhibition of the carbachol-induced contraction of rat urinary bladder, 5-HMT MS showed a much longer and more potent effect than tolterodine tablets. The mean urination time of the rats in the 5-HMT MS group was significantly decreased (p < 0.05 or p < 0.01) to less than 2 weeks.