The study identifies the transcription factor Sp1 as a positive regulator of GSDME expression, which is crucial for pyroptosis. Sp1 directly interacts with the GSDME promoter at the −36 to −28 site, enhancing GSDME gene transcription. Knockdown or inhibition of Sp1 reduces GSDME expression, thereby suppressing chemotherapy drug-induced pyroptosis. The regulation process synergizes with STAT3 activity and antagonizes DNA methylation but has minimal effect on GSDMD-mediated pyroptosis or TNF-induced necroptosis. This finding reveals a new mechanism of GSDME expression regulation, which may provide a potential target for inflammatory diseases and cancer therapy.The study identifies the transcription factor Sp1 as a positive regulator of GSDME expression, which is crucial for pyroptosis. Sp1 directly interacts with the GSDME promoter at the −36 to −28 site, enhancing GSDME gene transcription. Knockdown or inhibition of Sp1 reduces GSDME expression, thereby suppressing chemotherapy drug-induced pyroptosis. The regulation process synergizes with STAT3 activity and antagonizes DNA methylation but has minimal effect on GSDMD-mediated pyroptosis or TNF-induced necroptosis. This finding reveals a new mechanism of GSDME expression regulation, which may provide a potential target for inflammatory diseases and cancer therapy.