<p>Sepsis, a devastating microbe-induced inflammatory response, culminates in multi-organ dysfunction, with pyroptosis mediated by the non-canonical inflammasome being a pivotal factor. The mouse Caspase-11, central to this pathway, is directly activated by cytoplasmic lipopolysaccharide (LPS). Although ubiquitination is known to tightly regulate the inflammatory response in pyroptosis, its role in modulating the non-canonical inflammasome remains enigmatic. In this study, we unveil that the E3 ubiquitin ligase Smurf1 is a critical negative regulator of the non-canonical inflammasome pathway. Smurf1 orchestrates K48-linked polyubiquitination of Caspase-11 at K245 and K247 residues, leading to its degradation via the 26S proteasome. This process is further amplified by ERK phosphorylation of Smurf1 at the S148 site. In parallel, Caspase-11 modulates Smurf1 protein content through cleavage. Notably, macrophage-specific Smurf1 deficiency exacerbates sepsis-induced mortality in mice, attributed to the hyperactivation of the non-canonical inflammasome. Conversely, targeted supplementation of Smurf1 in macrophages mitigates the high mortality and inflammatory response associated with sepsis. Thus, Smurf1 emerges as a key player in modulating the activation of the non-canonical inflammasome in response to Gram-negative bacterial infections.</p>