Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) generally hijacks the cellular machinery of host cells for survival. However, how SARS-CoV-2 employs the host's deubiquitinase to facilitate virus replication remains largely unknown. In this study, we identified the host deubiquitinase USP22 as a crucial regulator of the expression of SARS-CoV-2 nucleocapsid protein (SARS-CoV-2 NP), which is essential for SARS-CoV-2 replication. We demonstrated that SARS-CoV-2 NP proteins undergo ubiquitination-dependent degradation in host cells, while USP22 interacts with SARS-CoV-2 NP and downregulates K63-linked polyubiquitination of SARS-CoV-2 NP, thereby protecting SARS-CoV-2 NP from degradation. Importantly, we further revealed that sulbactam, an antibiotic, can reduce USP22 protein levels, eventually promoting the degradation of SARS-CoV-2 NP and . This study reveals the mechanism by which SARS-CoV-2-encoded NP protein employs host deubiquitinase for virus survival and provides a potential strategy to fight against SARS-CoV-2 infection.IMPORTANCESevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid protein (SARS-CoV-2 NP) plays a pivotal role in viral infection by binding to viral RNA, stabilizing the viral genome, and promoting replication. However, the interactions between SARS-CoV-2 NP and host intracellular proteins had not been elucidated. In this study, we provide evidence that SARS-CoV-2 NP interacts with the deubiquitinase USP22 in host cells, which downregulates SARS-CoV-2 NP ubiquitination. This reduction in ubiquitination effectively prevents intracellular degradation of SARS-CoV-2 NP, thereby enhancing its stability, marking USP22 as a potential target for antiviral strategies. Additionally, our findings indicate that sulbactam significantly decreases the protein levels of USP22, thereby reducing SARS-CoV-2 NP levels. This discovery suggests a novel therapeutic pathway in which sulbactam could be repurposed as an antiviral agent, demonstrating how certain antibiotics might contribute to antiviral treatment. This work thus opens avenues for drug repurposing and highlights the therapeutic potential of targeting host pathways to inhibit viral replication.