Here we aimed to correlate different molecular weights of hyaluronic acid (HA), 200, 800 and 1437 kDa, used to decorate poly(lactic-co-glycolic acid) (PLGA)-based nanoparticles (NPs), to their cell uptakes. NP internalization kinetics in CD44-overexpressing breast carcinoma cells were quantified, using healthy fibroblast cells as reference. Actually, NP uptake and selectivity by tumor cells were maximized for NPs HA 800 kDa, while being minimum for NPs HA1400 kDa. This unexpected result could be explained considering that the interaction between NPs and tumor cells is dictated by rearrangement and conformation of that segment of HA chain that actually protrudes from the NPs. Overall, results obtained in this work point at how HA molecular weight, is pivotal project parameter in NP formulation to promote active targeting in the CD44 overexpressing cancer cells. Display omitted •NPs based on PLGA were physically linked and decorated with HA.•NPs formulations were produced with HA at 200, 800 and 1437 kDa MW respectively.•Physical chemical characterizations confirmed polymer arrangement.•HA 800 kDa NPs uptake kinetics in CD44-overexpressing tumor cells were maximized.•HA MW arrangement on NP surface affect active targeting by CD44 cancer cells.