In this study, a physiologically based pharmacokinetic (PBPK) model was established for valacyclovir based on absolute expression quantity of hPEPT1 along the entire length of the human intestine and other reliable in vitro, in vivo observed data. The PBPK model-3 defined acyclovir as metabolite of valacyclovir and simulated the plasma concentration-time profiles of valacyclovir and acyclovir simultaneously. It was validated strictly by a series of observed plasma concentration-time profiles. The average fold error (AFE) and absolute average fold error (AAFE) values were all smaller than 2. Then, it was used to quantitatively evaluate the effect of hPEPT1, luminal degradation rate, drug release rate and gastric residence time on the oral absorption of valacyclovir and acyclovir. The PBPK model-3 suggests that mainly 75% of valacyclovir was absorbed by active transport of hPEPT1. The luminal degradation of valacyclovir in the upper intestinal lumen cannot be considered the only reason for its incomplete bioavailability. The plasma concentration-time profiles of valacyclovir and its metabolite acyclovir were not sensitive to dissolution rate faster than T85% = 120 min. Prolonged gastric residence time of sustained release tablet can improve the oral absorption of valacyclovir. All in all, the PBPK model-3 in this study is reliable and accurate. It is useful for the research of clinical application and dosage forms design of valacyclovir.