In recent years evidence has been mounting for a role for mesenchymal stem cells (MSCs) in immunomodulation, anti-inflammatory processes, and paracrine signaling via secreted extracellular vesicles. In order to exploit these biological functions, systems to efficiently deliver genetic material into MSCs would therefore be highly desirable. In this study, efficient silencing of GFP expression by combining high N/P ratio siRNA and branched PEI (bPEI) polyplexes (siRNA-bPEI) polyplexes with glycosaminoglycans (GAGs), namely hyaluronic acid (HA), chondroitin sulfate (CS) and heparin sulfate (HS), and human serum albumin (HSA) is reported. These quaternary systems were characterized using surface charge, size and morphology and applied to MSCs, which represent a challenge due to their typically low transfection efficiency. The quaternary polyplexes promoted efficient charge shielding and release of siRNA in the cytoplasm with reduced toxicity. A high silencing efficiency of >90% (i.e., less than 10% remaining GFP expression) was achieved with noticeably reduced cellular toxicity, especially with siRNA-bPEI polyplexes modified with HA and HA + HSA. In general addition of GAGs led to more compact polyplexes. Endocytosis studies point to improved endosomal escape at high N/P ratios as a reason for high transfection efficiency and a role for hyaluronic acid in the uptake mechanism likely via CD44 interactions. Co-localization studies showed the polyplexes are stable in the cytosol over time, which correlates with a proper disassembly and subsequent silencing of GFP. Furthermore, GAG containing polyplexes were frequently co-localized with the nucleus. These findings in sum suggest that PEI/HSA/GAG based quaternary polyplexes are promising as transfection agents for MSCs. STATEMENT OF SIGNIFICANCE: Since mesenchymal stem cells (MSCs) are recruited to the site of tissue repair and play a role in immunomodulation, anti-inflammatory processes, and paracrine signaling, they present an excellent target for genetic engineering. However, delivery of genetic material into MSCs is challenging. In this study, >97% silencing of constitutive green fluorescent protein expression in human MSCs (hMSCs) using high N/P ratio polyplexes of branched-PEI-siRNA incorporating glycosaminoglycan as a charge neutralizer and human serum albumin as co-complexing agent is demonstrated. In addition to possessing good cytocompatibility and excellent cytosolic stability; polyplexes incorporating GAGs also showed altered endocytic uptake, with incorporation of hyaluronic acid promoting caveolae-mediated entry. Our system highlights the importance of physiologically derived macromolecules in delivery of genetic material into hMSCs.