Abstract We investigated the incorporation of gemcitabine into a colloidal carrier based on the biodegradable and biocompatible poly(d,l-lactide-co-glycolide) (PLGA) to optimize its anticancer activity. Two synthesis techniques (double emulsion/solvent evaporation, and Flow Focusing®) were compared in terms of particle geometry, electrophoretic properties (surface charge), gemcitabine vehiculization capabilities (drug loading and release), blood compatibility, and in vitro antitumor activity. To the best of our knowledge, the second formulation methodology (Flow Focusing®) has never been applied to the synthesis of gemcitabine-loaded PLGA particles. With the aim of achieving the finest (nano)formulation, experimental parameters associated to these preparation procedures were analyzed. The electrokinetics of the particles suggested that the chemotherapy agent was incorporated into the polymeric matrix. Blood compatibility was demonstrated in vitro. Flow Focusing® led to a more appropriate geometry, higher gemcitabine loading and a sustained release profile. In addition, the cytotoxicity of gemcitabine-loaded particles prepared by Flow Focusing® was tested in MCF-7 human breast adenocarcinoma cells, showing significantly greater antitumor activity compared to the free drug and to the gemcitabine-loaded particles synthesized by double emulsion/solvent evaporation. Thus, it has been identified the more adequate formulation conditions in the engineering of gemcitabine-loaded PLGA nanoparticles for the effective treatment of tumours.