Acyclovir-loaded mucoadhesive microspheres (ACV-ad-ms) using Ethylcellulose as matrix and Carbopol 974P NF as mucoadhesive polymer were prepared for the purpose of improving the oral bioavailability of acyclovir. The morphological properties of the microspheres were studied by optical microscopy and scanning electron microscopy (SEM). Drug loading and encapsulation efficiency was determined using HPLC method. In vitro and in vivo mucoadhesion of the microspheres was evaluated. Eggshell membrane was found to have a potential use for in vitro mucoadhesion measurement in place of stomach mucosa. In vitro drug release profiles and oral bioavailability of acyclovir in rats were also investigated. The release of the drug was influenced markedly by the medium pH and the proportion of Carbopol incorporated in the microspheres. The result of mucoadhesion study showed prolonged residence time of ACV-ad-ms in rats' gastrointestinal tract. In pharmacokinetics study, relatively steady plasma drug concentrations were observed within 8 h after oral administration of ACV-ad-ms to rats. The AUC(0-t) and mean residence time (MRT) of ACV-ad-ms (6055.9 ng h/mL and 7.2 h) were significantly higher than that of ACV suspension (2335.6 ng h/mL and 3.7 h) (P<0.05), which indicated that the bioavailability of acyclovir was greatly improved due to the prolonged retention of ACV-ad-ms in gastrointestinal tract.