Drug-loaded nanoparticles (DLNPs) enhance intracellular drug accumulation through endocytosis and are widely used in chemotherapy research. Despite progress, the therapeutic efficacy of DLNPs remains limited due to the complexity of the in vivo environment. Increasing evidence shows that mechanical cues such as substrate stiffness and fluid shear stress, are closely associated with cellular endocytosis. Yet, the effects of extracellular fluid viscosity have been largely overlooked. In this study, we found that high-viscosity culture medium (8 cP) treatment inhibits the endocytosis of DLNPs in comparison with low-viscosity culture medium (0.7 cP), decreasing intracellular drug concentration and reducing chemotherapy efficacy. Specifically, viscous resistance increased F-actin and vinculin density, thereby enhancing the cellular elastic modulus and membrane tension. Enhanced F-actin and vinculin also promote Yes-associated protein (YAP) nuclear translocation, and the translocated YAP functions as a key effector sustaining this mechanically reinforced state. As a result, such mechanical reinforcement raised the energetic barrier required for macropinocytosis and clathrin-mediated endocytosis, making these processes more difficult to occur. Inhibition of YAP abolishes this state and enhances the endocytosis and chemotherapy efficacy that are inhibited by high viscosity. This study suggests that lowering viscosity or inhibiting YAP function may enhance DLNP efficacy, offering new insights into chemotherapy.