BACKGROUND: Pediatric Crohn's disease (CD) is a chronic inflammatory bowel disorder that poses significant health risks to children. Although the precise etiology of CD remains elusive, further exploration is needed to identify diagnostic biomarkers and therapeutic targets. METHODS: This study utilized single-cell and bulk RNA sequencing data derived from ileal and colonic biopsy samples to explore the molecular mechanisms and cell types associated with CD, as well as to pinpoint potential biomarkers and therapeutic targets. RESULTS: The results revealed a more pronounced alteration in both the quantity and functional state of neutrophils in the CD cohort compared to those with ulcerative colitis and healthy controls. Neutrophils were present in higher proportions in the CD group, primarily in an activated state, potentially correlating with the presence of deep ulcerations and inflammatory histopathological features. Additionally, neutrophil interactions with other cell types were markedly enhanced in the CD group, making neutrophils the dominant participants in cell-to-cell communications. Further analysis indicated a shift in neutrophil phenotype from pro-inflammatory and antimicrobial to tissue-repairing, which may contribute to the progression and exacerbation of CD. CONCLUSION: IL1B, ICAM1, CXCL1, and CXCL9, primarily expressed in neutrophils, were potential biomarkers for CD. Neutrophils might be considered a potential target for pediatric CD. IMPACT STATEMENT: This study demonstrated that patients with CD exhibited a greater proportion of activated neutrophils, with enhanced interactions between neutrophils and all other cell types, resulting in neutrophils contributing the most cell-cell interactions within the CD gut. Neutrophils in the CD gut transition from a pro-inflammatory and antibacterial phenotype to one that promotes tissue healing, potentially influencing the progression and exacerbation of CD. Neutrophils represent a promising therapeutic target in pediatric CD. Hub genes associated with CD, including IL1B, ICAM1, CXCL1, and CXCL9, are predominantly expressed in neutrophils, positioning them as promising diagnostic biomarkers for CD.