Background: Intestinal ischemia/reperfusion (I/R) is a common clinical problem that occurs during various clinical pathological processes. Dexmedetomidine (DEX), a widely used anesthetic adjuvant agent, can induce protection against intestinal I/R in vivo; however, the underlying mechanism is not fully understood. In the present study, we aimed to investigate the protective effects of DEX and examine whether its mechanism was associated with the TLR4/MyD88/NF-κB signaling pathway. Methods: Sprague–Dawley rats were pretreated with DEX and then subjected to I/R-induced intestinal injury. In vivo, intestinal histopathological examination and scoring were performed, the levels of serum intestinal fatty acid-binding protein (I-FABP), intestinal tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and expression levels of TLR4, MyD88, and NF-κB in the intestine were determined. In in vitro experiments, the human colon carcinoma cell line (Caco-2) was incubated with DEX before deprivation/reoxygenation (OGD/R) treatment. The cell viability of Caco-2 cells, the levels of lactate dehydrogenase (LDH), TNF-α, and IL-1β in the supernatant, as well as protein expression of TLR4, MyD88, and NF-κB in Caco-2 cells, were measured. Statistical analysis was performed using SPSS version 21.0. Results: DEX preconditioning significantly reduced the intestinal pathological Chiu's score, serum I-FABP, intestinal TNF-α, IL-1β levels, and the protein expression of TLR4, MyD88, and NF-κB in the rats with intestinal I/R injury. Similarly, in vitro, DEX pretreatment protected against OGD/R-induced Caco-2 cell damage and inhibited TLR4/MyD88/NF-κB signaling, as evidenced by increased cell viability, decreased LDH activity, reduced TNF-α and IL-1β levels, as well as downregulated TLR4, MyD88, and NF-κB protein levels. Conclusions: Our findings suggested that DEX could reduce intestinal I/R injury in rats and OGD/R damage in Caco-2 cells, and this protection might be attributed to antiinflammatory effects and inhibition of the TLR4/MyD88/NF-κB signaling pathway. © 2020 Elsevier Inc.