Neonatal necrotizing enterocolitis (NEC) is a serious disease of premature infants that necessitates intensive care and frequently results in life-threatening complications and high mortality. Dedifferentiated fat cells (DFATs) are mesenchymal stem cell-like cells derived from mature adipocytes. DFATs were intraperitoneally administrated to a rat NEC model, and the treatment effect and its mechanism were evaluated. The NEC model was created using rat pups hand fed with artificial milk, exposed to asphyxia and cold stress, and given oral lipopolysaccharides after cesarean section. The pups were sacrificed 96 h after birth for macroscopic histological examination and proteomics analysis. DFATs administration significantly improved the survival rate from 25.0 (vehicle group) to 60.6% (DFAT group) and revealed a significant reduction in macroscopical, histological, and apoptosis evaluation compared with the vehicle group. Additionally, the expression of C–C motif ligand 2 was significantly decreased, and that of interleukin-6 decreased in the DFAT group. DFAT administration ameliorated 93 proteins mainly related to proteins of fatty acid metabolism of the 436 proteins up-/down-regulated by NEC. DFATs improved mortality and restored damaged intestinal tissues in NEC, possibly by improving the abnormal expression of fatty acid-related proteins and reducing inflammation.