The work herein presented reports the development of fucoidan/chitosan nanoparticles (NPs) loaded with gemcitabine and functionalized with ErbB-2 antibody at their surface (NPs + Gem + Ab). The maximum immobilization of ErbB-2 on NPs' surface was set at 10 μg mL-1 and resulted in NPs with a size around 160 nm, a polydispersity index of 0.18, and a zeta potential of 21 mV. ErbB-2 is overexpressed in some subtypes of breast cancers, and the targeting capability of the NPs + Gem + Ab system was confirmed by an increased cellular uptake of SKBR3 cells (ErbB-2 positive) when compared to MDA-MB-231 (ErbB-2 negative). To validate the targeting efficacy of NPs + Gem + Ab, a co-culture system with human endothelial and SKBR3 cells was established. Cytotoxic effects over endothelial cells were similar for all the tested conditions (between 25 and 30%). However, the NPs + Gem + Ab system presented increased toxicity over breast cancer cells, above 80% after 24 h, when compared to free Gem and NPs + Gem (around 15% and 20%, respectively). In vivo studies demonstrated that the developed targeting system significantly reduced tumor growth and the appearance of lung metastasis compared to untreated controls. In summary, the efficacy of the NPs + Gem + Ab system to target cancer cells was established and validated both in vitro and in vivo, being a compelling alternative strategy to current chemotherapeutic approaches.