Citrus Medica limonumessential oil (LEO) has been reported to have antibacterial and anti-inflammatory activities, but its protective effect in the intestine remains unknown. In this study, we researched the protective effects of LEO in relation to intestinal inflammation induced byE. coliK99. The mice were pretreated with 300, 600, and 1200 mg/kg LEO and then stimulated withE. coliK99. The results showed thatE. coliK99 caused immune organ responses, intestinal tissue injury, and inflammation. LEO pretreatment dose-dependently alleviated these changes by maintaining a low index in the thymus and spleen and producing a high content of immunoglobulin A, G, and M (IgA, IgG, and IgM) and low content of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6). Intestinal integrity as a consequence of the LEO pretreatment may be related to the high mRNA expression of intestinal trefoil factor (ITF) and the low mRNA expression of transforming growth factor-β1 (TGF-β1). Conclusively, an LEO pretreatment can alleviateE. coliK99-induced diarrhea, immune organ response, and body inflammation in mice by reducing the levels of inflammatory cytokines and improving the levels of immunoglobulin, and the intestinal integrity remained highest when maintaining the high mRNA expression of ITF and keeping the mRNA expression of TGF-β1 low in the intestinal tissue.