Purpose: Acute normovolemic hemodilution (ANH) is an autologous transfusion method, using blood collected during surgery, to reduce the need for allogeneic blood transfusion. ANH is controversial because it may lead to various complications. Among the possible complications, anastomotic leakage is one that would have a significant effect on the operation outcome. However, the relationship between ANH and anastomotic site healing requires additional research. Therefore, we conducted this prospective study of ANH, comparing it with standard intraoperative management, undergoing gastric anastomosis in rats. Methods: Sixteen Sprague-Dawley rats were randomly assigned to three groups: group A, surgery with ANH; group N, surgery with standard intraoperative management; and group C, sham surgery with standard intraoperative management. ANH was performed in group A animals by, removing 5.8-6.6 mL of blood and replacing it with 3 times as much crystalloid. All rats were enthanized on postoperative day 6, and histopathologic analyses were performed. Results: The mean hematocrit values, after hemodilution were 22.0% (range, 18.0%-29.0%), group A; 33.0% (29.0%-35.0%), group N; and 32.5% (29.0%-34.0%), group C. There were significant differences between groups A and N (P = 0.019, P = 0.009, P = 0.004, P = 0.039, and P = 0.027), and between groups N and C (P = 0.006, P = 0.027, P = 0.04, P = 0.008, and P = 0.009) with respect to inflammatory cell numbers, neovascularization, fibroblast numbers, edema and necrosis, respectively; there were no differences between groups A and N. Conclusion: In rat model, anastomotic complications did not increase in the ANH group, compared with the standard intraoperative management group.