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Gold nanoparticles (GNPs) are intended for use within a variety of biomedical applications due to their physicochemical properties. Although, in general, biocompatibility of GNPs with immune cells such as macrophages and dendritic cells is well established, the impact of GNPs on B lymphocyte immune function remains to be determined. Since B lymphocytes play an important role in health and disease, the suitability of GNPs as a B cell-targeting tool is of high relevance. Thus, we provide information on the interactions of GNPs with B lymphocytes. Herein, we exposed freshly isolated human B lymphocytes to a set of well-characterized and biomedically relevant GNPs with distinct surface (polyethylene glycol (PEG), PEG/poly(vinyl alcohol) (PEG/PVA)) and shape (spheres, rods) characteristics. Polymer-coated GNPs poorly interacted with B lymphocytes, in contrast to uncoated GNPs. Importantly, none of the GNPs significantly affected cell viability, even at the highest concentration of 20 μg/mL over a 24 h suspension exposure period. Furthermore, none of the nanosphere formulations affected the expression of activation markers (CD69, CD86, MHC II) of the naive B lymphocytes, nor did they cause an increase in the secretion of pro-inflammatory cytokines (i.e., IL-6, IL-1β). However, the absence of polymer coating on the sphere GNPs and the rod shape caused a decrease in IL-6 cytokine production by activated B lymphocytes, suggesting a functional impairment. With these findings, the present study contributes imperative knowledge toward the safe-by-design approaches being conducted to benefit the development of nanomaterials, specifically those as theranostic tools.