Background Activation of intestinal macrophages is implicated in the pathogenesis of neonatal necrotizing enterocolitis (NEC), yet its precise mechanisms remain unclear. Objective The purpose of this study is to investigate the role of macrophages and TNF-alpha via an inflammatory MicroRNA in NEC. Materials and methods Immunofluorescence (IF) staining of CD68, iNOS, and Arg-1 was employed to identify phenotypes of macrophage in the intestines of NEC infants and NEC mice. Expression of TNF-alpha, c-kit, and miR-222 was evaluated by qRT-PCR, Western blot, and immunochemical staining from the tissue samples. Results Large number of M1 macrophage infiltration was found in the NEC intestines. Expression of CD68, iNOS, and TNF-alpha were significantly increased, while c-kit was decreased distinctly in the NEC group. In the early phase of NEC mouse model, inhibition of M1 macrophages reduced the incidence of NEC and intestinal inflammation. We found that TNF-alpha upregulated the expression of miRNA-222 and inhibited the expression of c-kit. Conversely, such decrease of c-kit expression could be reversed by miR-222 antagonists. Furtherly, dual-luciferase assay confirmed that c-kit can be inhibited by miR-222 directly. Conclusion Macrophages activation in NEC intestine results in an increased inflammatory response and TNF-alpha production, accompanied with miR-222 upregulation and c-kit suppression. Modulations of M1 macrophages, TNF-alpha or miR-222 may be potential therapeutic targets for NEC treatment.