The absorption-enhancing effect of the simultaneous administration of sodium caprate (Cap-Na) and dipotassium glycyrrhizinate (Grz-K) was investigated to clarify an effect of Grz-K. A combination of 0.1% (w/v) Cap-Na and 2% (w/v) Grz-K had a rapid and long-lasting absorption-enhancing activity in Caco-2 cell monolayers under conditions where Cap-Na and Grz-K showed a weak and no activity, respectively. The simultaneous treatment of a Caco-2 cell monolayer with Cap-Na and Grz-K showed no change in intracellular calcium ion level, although a major mechanism of absorption-enhancing effect for Cap-Na was elevation of intracellular calcium ion level. On the other hand, the simultaneous enhancing effect of Cap-Na and Grz-K was inhibited by H7, a PKC inhibitor. Possibly, Grz-K showed an absorption-enhancing effect via PKC cellular signaling pathway after penetration into cell according to increasing membrane permeability by Cap-Na. The absorption of sCT by the rat colon was enhanced by a combination of 0.1% (w/v) Cap-Na and 2% (w/v) Grz-K, and its effect continued even 9h after the onset of the experiment. Furthermore, the simultaneous treatment of 0.1% (w/v) Cap-Na and 2% (w/v) Grz-K showed a negligible histological changes to the colon mucosal membrane and a negligible toxicity on Caco-2 cell monolayer. A combination of Cap-Na and Grz-K shows a synergistic absorption-enhancing effect with little mucosal injury, which is applicable to colon-specific delivery.