Background: Our aim was to evaluate in vitro perfusion of the rat small intestine as a method to study the pathophysiology of hypoperfusion and to minimize its adverse effects. Methods: Rat small intestine was perfused for 24 h using RPMI + albumin for the lumen and RPMI + erythrocyte concentration (Hct 20%) in the vasculature, at 22°C, 15°C, 10°C, and 6°C. The outcome was evaluated by histologic grading and permeability for polyethylene glycol 900 (PEG 900). Results: Perfusion of the intestine at 6°C resulted in lower degree of mucosal damage and permeation of PEG 900, compared with storage and perfusion at higher temperatures. Conclusions: In vitro perfusion of the small intestine, using histologic grading of mucosal lesions and permeation of PEG 900 as indicators, is one method that can be applied to study the pathophysiology of intestinal hypoperfusion. Perfusion at 6°C significantly reduced the level of damage to the intestinal mucosa.