<p>Neonatal necrotizing enterocolitis (NEC) is a life-threatening gastrointestinal disease of premature infants, characterized by immune dysregulation and compromised intestinal barrier integrity. Interleukin-27 receptor α (IL-27Ra), a critical component of the JAK-STAT signaling pathway, exhibits dual pro- and anti-inflammatory roles in various inflammatory conditions. However, its role in NEC pathogenesis remains unclear. To elucidate the functional role of IL-27Ra in NEC development and assess its potential as a therapeutic target. A multi-tiered approach was employed, including integrative analysis of clinical NEC specimens by single-cell and bulk RNA sequencing, and a neonatal mouse NEC model. NEC was induced in mice via hyperosmolar formula feeding combined with LPS gavage, intermittent hypoxia, and cold stress. Additional experiments included immunofluorescence staining for IL-27Ra, cytokine profiling (ELISA, quantitative real-time PCR (qPCR)), use of IL-27Ra knockout (IL-27Ra^-/-) mice, and histopathological scoring of intestinal injury. In NEC patient intestinal tissues, IL-27Ra expression was significantly upregulated in immune cells, with expression levels positively correlating with pro-inflammatory mediators (e.g., IL-6) and inversely correlating with barrier-associated proteins (e.g., TJP1). In the neonatal mouse NEC model, genetic ablation of IL-27Ra (IL-27Ra^-/-) led to reduced histopathology scores, decreased IL-6 production (ELISA and qPCR), and restored tight junction protein expression (TJP1, OCLN). IL-27Ra promotes NEC by amplifying intestinal inflammation and damaging the mucosal barrier. Thus, IL-27Ra is identified as a promising therapeutic target. Pharmacological blockade of IL-27Ra signaling may provide a dual benefit in NEC—mitigating excessive inflammation while restoring barrier integrity. These findings are primarily derived from NEC mouse models and await clinical validation.</p>