Adjuvants are substances that increase the immune response to a given antigen. In the development of novel vaccine adjuvants/systems, saponins are one of the most attractive molecules due to their altered immunomodulatory activities. In this study, we tried to develop PEG (polyethylene glycol)/cholesterol-based lipid nanoparticles (LNPs) to deliver the Astragaloside VII (AST-VII) and potentiate adjuvant properties of AST-VII for the influenza vaccine. In the formation of PEG/cholesterol/AST-VII-based LNPs (PEG300: Chol-AST-VII LNPs), 3 different primary solvents (acetone, ethanol, and chloroform) were evaluated, employing their effects on hydrodynamic particle size, distribution, surface chemistry, and colloidal stability. Prepared nanoparticles were simply admixtured with inactivated influenza antigen (H3N2) and applied to PMA (phorbol 12-myristate 13-acetate)-ionomycin treated human whole blood to evaluate their cytokine release profile. PEG300: Chol-AST-VII LNPs (80.2 ± 7.7 nm) were obtained using chloroform as a desolvation agent. Co-treatment of PMA-ionomycin with AST-VII and PEG300: Chol-AST-VII LNPs significantly increased the levels of IL-2 and IFN-g, compared to PMA-ionomycin alone. In the presence of H3N2, AST-VII was able to augment IL-17A, while PEG300: Chol-AST-VII LNPs stimulated the production of IFN-g. Hemolysis was only observed in PEG300: Chol-AST-VII LNPs (250 μg/mL) treatment. AST-VII and AST-VII-integrated LNPs could be used as efficacious adjuvants for an inactivated H3N2 vaccine in vitro, and cytokine response through Th1/Th17 route was reported. Copyright © 2020 The Author(s).