Gemcitabine is one type of nucleoside drugs which be used for the treatment of tumours in the clinical. But the clinical trial indicate that gemcitabine has low intestinal membrane permeability and its oral bioavailability is nearly about 37.6% in vivo. PEPT 1 is one of the intestina peptide transporter,it can notablely improved intestinal membrane permeability of peptidomimetic compounds in vivo.In this study,8 different types of gemcitabine 3′-amino acid ester peptidomimetic compounds were regioselective synthesized, the structure of the target compound was confirmed by means of ESI-MS and 1HNMR. The biological activity assay results showed that all the compounds affinity with PEPT 1 and their permeability across Caco-2 cells were obviously ameliorated compared with gemcitabine, and compounds 5c,5e also remained apparente activity to inhibit the proliferation of NCl-H460 cells.