Neutrophils are key players in the hyperinflammatory response during SARS-CoV-2 infection, exhibiting features of deregulation and reprogramming. The cytosolic proliferating cell nuclear antigen (PCNA) is a scaffolding protein highly dependent on the microenvironment status and known to interact with numerous proteins that regulate neutrophil survival, NADPH oxidase-dependent reactive oxygen species (ROS) production, and cell energy metabolism. In this sense this study aimed to examine the cytosolic protein content and PCNA interactome in neutrophils from COVID-19 patients. Methods: Proteomics analysis was performed on neutrophil cytosols from healthy donors and patients with severe or critical COVID-19. In vitro approaches with ultrapure neutrophils were employed to validate the role of PCNA dynamics in neutrophil overactivation during COVID-19.