Chinese hamster ovary (CHO) cells are widely used for the production of recombinant biologics, but their application in transient gene expression (TGE) systems remains limited by reduced efficiency at high cell density, known as the cell density effect (CDE). Increasing evidence suggests that extracellular factors, including extracellular vesicles (EVs), contribute to this limitation. However, their role in CHO-based TGE systems remains poorly understood. In this study, we investigated the impact of EV accumulation and depletion on transfection efficiency, virus-like particle (VLP) production, and cellular responses in CHO cells. EV production increased approximately 4.2-fold at high cell density and was influenced by extracellular conditions, including media replacement. EV depletion partially restored transfection efficiency and productivity to approximately ∼60% of low cell density levels. In addition, moderate overexpression of UDP-glucose ceramide glucosyltransferase (UGCG) further enhanced transfection efficiency and VLP productivity, reaching levels comparable to low cell density conditions when combined with fresh media replacement. Transcriptomic and proteomic analyses showed that EV depletion induces a multi-layered cellular response, including transient activation of proteostasis pathways, sustained upregulation of vesicle trafficking, and later activation of lysosomal and autophagy-related processes. These responses suggest that EV removal perturbs extracellular homeostasis and triggers adaptive intracellular reprogramming. Overall, these findings demonstrate that EVs play a dual role in CHO TGE systems, acting both as inhibitory extracellular components and as regulators of cellular homeostasis. Modulating extracellular conditions in combination with targeted manipulation of lipid metabolism can partially alleviate the CDE but also introduces stress-related trade-offs that must be considered in process design.