The effects of a series of pharmaceutical excipients, including Span 80, Brij 30, Tween 20, Tween 80, Myrj 52, and sodium lauryl sulfate (with increasing hydrophilic-lipophilic balance (HLB) values) on the intracellular accumulation, transport kinetics, and intestinal absorption of epirubicin were investigated in both the human colon adenocarcinoma (Caco-2) cell line and the everted gut sacs of rat jejunum and ileum. The possible use of these excipients as multidrug resistance (MDR) reversing agents also was examined. Epirubicin uptake experiments using a flow cytometer showed that these selected excipients markedly enhanced the intracellular accumulation of epirubicin in Caco-2 cells in a dose-dependent manner. The optimal effect on the epirubicin uptake was characteristic of excipients with intermediate HLB values ranging from 10 to 17. Moreover, the optimal net efficacy was observed for excipients with polyoxyethylene chains and intermediate chain length of fatty acid and fatty alcohol (monolaurate for Tween 20, monooleate for Tween 80, monostearate for Myrj 52, and lauryl alcohol for Brij 30). These excipients significantly increased apical to basolateral absorption and substantially reduced basolateral to apical efflux of epirubicin across Caco-2 monolayers. Furthermore, the addition of Tween 20, Tween 80, Myrj 52, and Brij 30 markedly enhanced mucosal to serosal absorption of epirubicin in the rat jejunum and ileum. This study suggests that inhibition of intestinal P-glycoprotein (P-gp), multidrug resistance associated protein family (MRPs), or other transporter proteins by pharmaceutical excipients may improve oral absorption of drugs in MDR spectrum. The optimal HLB values of surfactant systems with suitable hydrocarbon chains and polar groups are an important factor in designing promising epirubicin formulations for reversing MDR. In conclusion, therapeutic efficacy of epirubicin may be enhanced by the use of such low toxicity excipients as absorption enhancers and MDR modulators in formulations. This provides a potential strategy for improving bioavailability in the optimization of formulations for drugs performing intestinal absorption and secretion.