Gemcitabine, fin anticancer nucleoside analogue, undergoes rapid enzymatic degradation following intravenous injection. This necessitates the administration of a high order of doses to observe a required therapeutic response, while such high doses result in significant side effects. To improve the intravenous delivery of gemcitabine and simultaneously enhance its antitumor activity, we have investigated its incorporation into a drug nanoplatform based on the biodegradable polymer chitosan. Two gemcitabine loading methods have been investigated: (i) entrapment into the polymeric network (entrapment procedure): drug incorporation prior to the coacervation process that leads to the formation of gemcitabine-loaded chitosan (GemChit) nanoparticles; and (ii) surface deposition onto already formed chitosan nanoparticles after incubation in gemcitabine solution (adsorption procedure). The former method produced much higher gemcitabine loading values and a sustained release profile. The main factors determining the gemcitabine loading and release kinetic have also been analyzed. Following intravenous injection, the Gem Chit formulation displayed a significantly improved antitumor activity comparatively to free gemcitabine, which was further confirmed by histology and immunohistochemistry studies, suggesting the potential of this chitosan-based gemcitabine nanomedicine for the effective treatment of tumors.