A doubly enteric-coated multiple-unit tablet (DET) of bisacodyl (BD) was formulated to selectively deliver the stimulant laxative to the large intestine. Solubilized BD in surfactants was adsorbed into the porous carrier and primarily coated with different combinations of pH-sensitive polymers (Eudragit S and Eudragit L) and time-dependent release polymer (Eudragit RS). BD-loaded granules were compressed into tablets and coated again with pH-sensitive polymers (Eudragit S:Eudragit L = 1:1). The multiple-unit tablet was optimized with respect to the granular coating compositions (Eudragit S:Eudragit L:Eudragit RS = 5:1:4) and coating level (12.5%), and coating level on the tablet (25%), by evaluating in vitro release profile in continuous dissolution medium. Drug release from the optimized tablet was effectively retarded in the simulated gastric and small intestinal fluids (below 7%), but profound drug liberation was attained in the colonic fluid (over 50%). On the other hand, drug release from the marketed product (Dulcolax®, Boehringer Ingelheim Pharma), a reference drug, in the gastric and small intestinal fluids was reached to 30%, while that in the colonic fluid was only 7%. In an in vivo efficacy study in loperamide-induced constipated rabbits, a remarkable recovery in fecal secretion was observed in the DET-treated group 24 h post-dosing, compared to vehicle-treated (p < 0.05) and the marketed product-treated groups (p < 0.05). Moreover, pharmacokinetic evaluation in the constipated rabbits revealed that the DET system significantly lowered the systemic exposure compared with the marketed product (p < 0.05), by hindering drug release in the upper intestine, a preferential absorption site. Therefore, the novel colon-targeted delivery system may be an alternative for boosting pharmacological responses in the colon, while diminishing the intestinal irritation and/or systemic adverse effect of the stimulant laxative.