Pyroptosis is a kind of programmed cell death, which is characterized by the activation of inflammatory caspase and the cleavage of gasdermin proteins. It is widely involved in the occurrence and development of tumors. Studies have shown that ubiquitin related proteins play a key regulatory role in many biological processes. However, the role and molecular mechanism of ubiquitin-related proteins in pyroptosis have not been well identified. Here, using CRISPR-Cas9 screening, we identified a deubiquitinating enzyme (USP48) that has the most significant regulatory effect on cell pyroptosis. USP48 stabilizes GsderminE (GSDME) expression by causing deubiquitination of it, thereby achieving its regulatory effect on pyroptosis. USP48 prevents the degradation of GSDME by inhibiting K63-linked ubiquitination at positions K120 and K189 of GSDME. Clinical tissue testing confirmed that the expression of USP48 has a significant positive correlation with GSDME and pyroptosis-related factors. GSDME plays a crucial role in the regulation of cell pyroptosis by USP48. The single-cell sequencing results showed that the functions of T cells and tumor-associated macrophages in the tumor microenvironment are inhibited to varying degrees after USP48 gene knockout. Finally, the tumor formation experiment in mice confirmed that overexpression of USP48 could effectively improve the therapeutic effect of PD-1 inhibitors. These findings define a pyroptosis regulation pathway and indicate that activation of the pharmacological activity of USP48 may provide an effective strategy to sensitize cancer cells to pyroptosis-related immunotherapeutic resistance.