Raltitrexed (RTX) has shown clinical activity in a variety of advanced solid tumours. Its oral bioavailability is low and its intestinal absorption mechanism is not clear. In the present study, the absorption mechanism of RTX in the small intestine was investigated, and the effects of absorption enhancers and efflux transporter inhibitors were evaluated by in vitro transport studies using the Caco-2 cell model and in situ perfusion experiments in rats. Oral bioavailability of RTX in rats in the presence or absence of enhancers were also investigated. The results of in vitro and in situ experiments indicated that the kinetic model of combined mechanism (active and passive transport) fitted the concentration-time data of RTX best with the highest R2 and lowest SSE (Sum of Squares for Error). The apparent or effective permeability coefficient (P(app) or P(eff)) of RTX remained statistically constant in a certain concentration range, then decreased when the concentration increased. But the decrease trend did not continue with further increase in concentration. And folic acid could competitively inhibit RTX absorption. These results suggested that a combined absorption mechanism for RTX existed. Furthermore, within certain concentration ranges, Carbomer 934P and sodium caprate (Cap-Na) exhibited significant absorption enhancement effects with low toxicity, whereas the enhancement effects of sodium deoxycholate (Deo-Na) were accompanied with acute toxicities. Moreover, probenecid and pantoprazole obviously enhanced RTX absorption, demonstrating that RTX is a substrate of the multidrug resistance protein (MRP) and breast cancer resistance protein (BCRP). A secretion experiment indicated that RTX could be effluxed into the intestines both with bile and by active efflux action. Oral bioavailability of RTX was significantly improved by the investigated absorption enhancers and transporter inhibitors, which is consistent with the in vitro and in situ experiments.