Necrotizing enterocolitis (NEC) is a severe inflammatory gastrointestinal disorder affecting preterm infants. Through fecal metabolomics, we found linoleic acid compounds (LAs) reduced in NEC and negatively correlated with C-reactive protein and NEC Bell stage. Conjugated linoleic acid (CLA) is a special kind of LAs with multiple functions. This study aims to investigate the role and mechanism of CLA in vivo and vitro. Experimental results in neonatal rats demonstrated that CLA significantly reduced mortality, weight loss, improved pathological scores, relieved inflammation and preserved intestinal cell homeostasis. In vitro, CLA promoted cell proliferation and reduced cell injury. RNA-sequencing analysis indicated that PPAR signaling pathway might be critical. Experiments showed that CLA facilitated PPARγ/NFκB signaling pathway by upregulating PPARγ expression, inactivating phosphorylated NFκB, and preventing translocation of NFκB P65. This study underscores the therapeutic potential of CLA and provides new insight and direction for developing NEC interventions.