Background and purpose: Intestinal ischemia-reperfusion (I/R) injury is a common clinical issue involving sepsis, shock, necrotizing enterocolitis, mesenteric thrombosis. Inflammatory reactions and cellular apoptosis are main involved processes in intestinal I/R injury. Data regarding the effect of thymosin beta 4 (Tβ4) on intestinal I/R injury are scarce. It was designed to evaluate the potential effects of Tβ4 on intestinal IR injury Methods: The study was conducted on 3 groups of adult male albino rats: each containing ten rats: group I (I/R group), group II (Sham- operated group), and group III (Tβ4-treated group). After one hour of intestinal ischemia, reperfusion was performed by releasing the clamp. Thirty minutes before reperfusion, the rats in group III received subcutaneous injections of 30 mg Tβ4 per kg and 0.1 ml physiologic saline. Rats in groups I and II received only 0.1 ml physiologic saline. Intestinal histopathologic examination and scoring the degree of injury were done according to chiu's score. Biochemical analyses of malondialdehyde (MDA), super oxide dismutase (SOD) and glutathione peroxidase (Gpx) levels in addition to TNF-α, IL-6 and caspase-3 levels in the rat intestinal tissues were determined. Serum and tissue nitric oxide (NO) levels were also measured. Results: Intestinal I/R injury was confirmed by intestinal histopathologic examination. In the I/R group, tissue MDA, TNF-α, IL-6 and caspase-3 levels were significantly elevated, however, tissue SOD, Gpx, serum and tissue NO levels were significantly declined when compared to the sham group. On receiving Tβ4, the findings demonstrated significant reductions of tissue MDA, TNF-α, IL-6 and caspase-3 levels and significant elevations of serum and tissue NO, tissue SOD and Gpx levels compared to non-treated one. Further, rats treated with Tβ4 showed amelioration of the degree of intestinal I/R lesions and improvement of intestinal score injury compared to non-treated group. Conclusions: Tβ4 administration significantly protected the intestinal tissues against the intestinal I/R injuries. Tβ4 treatment appears to be a promising protective and therapeutic approach for intestinal I/R injury.