Gemcitabine is a widely used chemotherapeutic drug that is administered via intravenous infusion due to a low oral bioavailability of only 10%. This low oral bioavailability is believed to be the result of gemcitabine's low intestinal permeability and oral absorption, followed by significant presystemic metabolism. In the present study, we sought to define the mechanisms of gemcitabine intestinal permeability, the potential for saturation of intestinal uptake, and the transporter(s) responsible for mediating the oral absorption of drug using in situ single-pass intestinal perfusions in mice. Concentration-dependent studies were performed for gemcitabine over 0.5-2000 μM, along with studies of 5 μM gemcitabine in a sodium-containing buffer ± thymidine (which can inhibit concentrative (i.e., CNT1 and CNT3) and equilibrative (i.e., ENT1 and ENT2) nucleoside transporters) or dilazep (which can inhibit ENT1 and ENT2), or in a sodium-free buffer (which can inhibit CNT1 and CNT3). Our findings demonstrated that gemcitabine was, in fact, a high-permeability drug in the intestine at low concentrations, that jejunal uptake of gemcitabine was saturable and mediated almost exclusively by nucleoside transporters, and that jejunal flux was mediated by both high-affinity, low-capacity (Km = 27.4 µM, Vmax = 3.6 pmol/cm2/s) and low-affinity, high-capacity (Km = 700 µM, Vmax = 35.9 pmol/cm2/s) transport systems. Thus, CNTs and ENTs at the apical membrane allow for gemcitabine uptake from the lumen to enterocyte, whereas ENTs at the basolateral membrane allow for gemcitabine efflux from the enterocyte to portal venous blood.