Abstract: The intestinal mucosal barrier serves as a vital guardian of the gut health, maintaining a delicate equilibrium between gut microbiota and host immune homeostasis. Gasdermin D (GSDMD), a key executioner of pyroptosis downstream of the inflammasome, has been found to play intricate roles in modulating colitis by influencing intestinal macrophages and regulating mucus secretion from goblet cells. However, the exact nature of the regulatory function of GSDMD in maintaining intestinal immune homeostasis and defending against pathogens remains to be elucidated. In the current study, by using the Citrobacter rodentium infection model, we found that GSDMD played a key role in the defense against intestinal Citrobacter rodentium infection, with high expression levels in intestinal epithelial and lamina propria myeloid cells. Our results showed that GSDMD acted specifically in intestinal epithelial cells to combat the infection, independently of its effects on antimicrobial peptides or mucin secretion. Instead, the resistance was mediated by the N-terminal fragment of GSDMD, highlighting its importance in intestinal immunity. However, the specific mechanism underlying the N-terminal activity of GSDMD in protecting against intestinal bacterial infections requires future investigation.