Intestinal ischemia and reperfusion (I/R) triggers a systemic inflammatory response characterized by leukocyte mobilization from the bone marrow, release of cytokines to the circulation, and increased microvascular permeability, leading to high mortality. Females have shown attenuated inflammatory response to trauma when compared with males, indicating a role for female sex hormones in this process. Here, we have evaluated the effect of estradiol on the local gut injury induced by I/R in male rats. I/R was induced by the clamping of the superior mesenteric artery for 45 min, followed by 2 h of reperfusion. A group received 17β-estradiol (280 μg/kg, i.v., single dose) at 30 min of ischemia. Morphometric analysis of the gut showed I/R induced a reduction of villous height that was prevented by estradiol. White blood cells, notably granulocytes, were mobilized from the circulation to the intestine by I/R, which was also prevented by estradiol treatment. Groups had the intestine wrapped in a plastic bag to collect intestinal fluid, where leukocytes count, TNF-α, and IL-10 levels were increased by I/R. Serum chemokines (CINC-1, MIP-1α, MIP-2), ICAM-1 expression in the mesenteric tissue, and neutrophils spontaneous migration measured in vitro were also increased after I/R. Estradiol treatment reduced leukocytes numbers and TNF-α on intestinal fluid, serum chemokine release and also downregulated MIP-1α, MIP-2 gene expression, and spontaneous in vitro neutrophil migration. In conclusion, estradiol blunts intestinal injury induced by I/R by modulating chemokines release and leukocyte trafficking.