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Highly purified adenovirus preparations are essential for fundamental research and therapeutic use. However, the impact of viral concentration during CsCl gradient ultracentrifugation on virion integrity is poorly understood, and this incomplete mechanistic understanding may adversely affect product quality and constrain further advances in research. In this study, we examined how viral input affects adenovirus infectivity, cytotoxicity, yield, and structural integrity using transmission electron microscopy and analysis tools. High-concentration preparations (AdV-HC) showed significant aggregation, capsid damage, and considerably lower TCID₅₀ values, while optimally concentrated preparations (AdV-OC) preserved intact icosahedral structures and high infectivity. Efforts to reverse aggregation failed, showing that excessive viral concentration during purification causes irreversible structural harm. These findings indicate that overloading during CsCl ultracentrifugation promotes vector aggregation, thereby compromising virion stability and biological activity. To preserve structural integrity and ensure consistent performance, we recommend maintaining the final purified vector concentration at or below 2 mg/mL. Based on this threshold, we defined theoretical optimal optical density (OD) ranges for adenovirus concentration in cell lysates prior to ultracentrifugation to minimize vector aggregation during purification. Therefore, this study provides a simple and effective strategy for improving the consistency, purity, and infectivity of adenovirus preparations for both research and clinical applications.