--- - |2 miR‐124 was a promoter of necrotizing enterocolitis (NEC), which promotes the intestine cell apoptosis and inflammatory cell infiltration through the inhibition of Toll‐like receptor 9 (TLR9) expression by targeting rho‐associated coiled‐coil‐containing protein kinase 1 (ROCK1). Abstract Backgrounds Necrotizing enterocolitis (NEC) was one of the main causes of morbidity and mortality in neonates. Our objective was to detect the mechanism of miR‐124 in small bowel tissues of NEC. Methods Hematoxylin and eosin (H&E) staining was used to detect the repair of the damaged tissues in rat NEC model. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was used to evaluate the cell apoptosis level in intestinal tissue. Reverse transcription polymerase chain reaction (RT‐PCR) was used to detect the messenger RNA (mRNA) expression level of miR‐124, Rho‐associated coiled‐coil‐containing protein kinase 1 (ROCK1), myosin phosphatase target subunit 1 (MYPT1), and Toll‐like receptor 9 (TLR9) in NEC tissues and IEC‐6 cells. Luciferase reporter assay was used to verify whether ROCK1 is a direct target of miR‐124. Results miR‐124 was overexpressed in the NEC tissues, while ROCK1 and MYPT1 was downregulated in the NEC tissues. Inhibition of miR‐124, suppressed the intestinal cell apoptosis and promoted the expression of ROCK1 and MYPT1. What is more, overexpression of miR‐124 could inhibit the expression of ROCK1, TLR9, and MYPT1. Luciferase assay confirmed that miR‐124 can regulate the transcriptional activity of ROCK1 through binding its 3′‐UTR region. Conclusion miR‐124 was a promoter of NEC, which promotes the intestine cell apoptosis and inflammatory cell infiltration through the inhibition of TLR9 expression by targeting ROCK1. - 'Journal of Cellular Physiology, EarlyView. '