The aim of this research is to clarify the influence of the viscosity of the nasal formulation on in vivo nasal drug absorption and its mechanism using an in vitro Caco-2 system. The drug solution was made viscous by the addition of dextran (Dex). The disappearance of FITC-labeled Dextran (FD, a marker of the dosing solution) applied with control solution followed monoexponential kinetics, while FD applied with Dex solution showed biexponential elimination. The mean residence time of FD in the nasal cavity was increased with the increase in Dex concentration. The nasal absorption of acyclovir was similar in the formulation with low viscosity, increased in the formulation with moderate viscosity and markedly decreased in the formulation with high viscosity. The result from the normal Caco-2 transport study could not explain the relation of in vivo drug absorption with viscosity, while the modified Caco-2 system provided data partly reflecting the change in in vivo absorption in rats. In conclusion, the residence of the applied solution in the nasal cavity was enhanced by the addition of Dex in a viscosity-dependent manner. Moderate viscosity of the dosing solution improved the in vivo nasal absorption of acyclovir, while higher viscosity decreased it.