Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), responsible for coronavirus disease 2019 (COVID-19), disrupts the alveolar epithelial barrier and exacerbates airway inflammation, leading to acute respiratory failure. The Envelope (E) protein is key to virulence, notably through its PDZ-binding motif (PBM), which interacts with host PDZ proteins, affecting signaling pathways and pathogenicity. This study investigates the PBM's role in virulence by generating PBM-deficient mutant viruses and assessing their impact in vitro and in vivo. The mutants showed delayed replication and reduced cytopathic effects in vitro. In vivo, infected hamsters exhibited less weight loss, lower viral loads, and reduced inflammation, indicating attenuated pathogenicity. Histological analysis confirmed milder airway damage. Additionally, PBM-deficient viruses had impaired interactions with tight junction proteins like ZO-1, a PDZ-containing protein essential for epithelial integrity. Although the PBM played a key role in airway pathology, its impact on neuroinvasion was minimal during the acute phase of infection. Thus, the E protein PBM plays a critical role in SARS-CoV-2's fitness, virulence, and pathogenicity, through the disruption of cell junctions and inflammation, underscoring its potential as a therapeutic target.