IRW (Ile-Arg-Trp), a bioactive peptide isolated from egg ovotransferrin, has been shown to exert anti-inflammatory effects. In this study, the effects of IRW on inflammatory cytokines and microbiota were explored in human umbilical vein endothelial cells (HUVECs) and a lipopolysaccharide (LPS)-induced rat model of inflammatory peritonitis. Rats were injected intraperitoneally with LPS to establish peritonitis. HUVECs were exposed to IRW for 12 h before introducing LPS. Notably, IRW exerted beneficial effects against LPS-induced peritonitis, specifically, by reducing the serum levels of tumour necrosis factor (TNF)-α and interleukin (IL)-6 and myeloperoxidase (MPO) activity (P<0.05). A faecal microbiota analysis revealed that IRW significantly increased the Shannon and decreased the Simpson indices (P<0.05). Furthermore, IRW treatment significantly inhibited the LPS-induced enhancement of TNF-α, IL-8, intercellular cell adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) expression in HUVECs (P<0.05). In conclusion, IRW supplementation inhibited the inflammatory mediator synthesis and LPS-induced inflammatory responses and influenced the gut microbiota.