Apicidin, a potential oral chemotherapeutic agent, possesses potent anti-histone-deacetylase activity. After oral administration, the total bioavailability of apicidin is known to be low (14.2%-19.3%). In the present study, we evaluated the factors contributing to the low bioavailability of apicidin by means of quantitative determination of absorption fraction and first-pass metabolism after oral administration. Apicidin was given to rats by five different routes: into the femoral vein, duodenum, superior mesenteric artery, portal vein, and carotid artery. Especially, the fraction absorbed (FX) and the fraction that is not metabolized in the gut wall (FG) were separated by injection of apicidin via superior mesenteric artery, which enables bypassing the permeability barrier. The FX was 45.9% ± 9.7%, the FG was 70.9% ± 8.1% and the hepatic bioavailability (FH) was 70.6% ± 12.3%, while the pulmonary first-pass metabolism was minimal (FL = 102.8% ± 7.4%), indicating that intestinal absorption was the rate-determining step for oral absorption of apicidin. The low FX was further examined in terms of passive diffusion and transporter-mediated efflux by in vitro immobilized artificial membrane (IAM) chromatographic assay and in situ single-pass perfusion method, respectively. Although the passive diffusion potential of apicidin was high (98.01%) by the IAM assay, the in situ permeability was significantly enhanced by the presence of the P-glycoprotein (P-gp) inhibitor elacrider. These data suggest that the low bioavailability of apicidin was mainly attributed to the P-gp efflux consistent with the limited FX measured in vivo experiment.