Objective To explore the intestinal absorption-promoting effect of volatile oils of Shichangpu, benzoin and storax on Rhodamine-123 (Rho-123) and its mechanisms. Methods Rat in vitro everted gut sac (EGS) model was made. The EGS was filled with 1.0 mL K-R solution, and suspended in 45.0 mL 37℃ K-R solution with or without 60 μg/mL volatile oils of Shichangpu, benzoin or storax. After incubation for 30 min, Rho-123 was added in the experimental solution outside the EGS. Rho-123 content in the EGS was determined by high-performance liquid chromatography-fluorescence detector. The human colon carcinoma cell lines Caco-2 were cultured in DMEM and incubated with 80 μg/mL volatile oils of Shichangpu, benzoin or storax for 48 h. P-glycoprotein (P-gp) expression in Caco-2 cells was assessed using flow cytometry, and P-gp gene MDR1 mRNA expression was determined by qPCR. Results The volatile oils of Shichangpu, benzoin and storax each significantly increased the absorption rate constant and apparent permeability coefficient of Rho-123 in rat jejunum and ileum (P<0.01), reduced P-gp expression and MDR1 mRNA level in Caco-2 cells (the decreased rates of P-gp expression were 53.15%, 55.10% and 61.86%, and of MDR1 mRNA levels were 55.41%, 16.24% and 38.46%, P<0.01). Conclusion Inhibiting the protein and MDR1 mRNA expressions of P-gp may be one of the main mechanisms of volatile oils of Shichangpu, benzoin and storax in promoting Rho-123 intestinal absorption.