Background: Liver failure/disease leads to hyperammonemia, a central component in the pathogenesis of hepatic encephalopathy. There is increasing evidence that oxidative stress exacerbates the neuropsychological effects of hyperammonemia in patients with liver disease, possibly by inducing brain edema. The latter represents a common entity in cirrhotic patients with hepatic encephalopathy. Since rats following 4-week portacaval anastomosis (PCA) become hyperammonemic but do not develop oxidative stress or brain edema, we aimed to investigate the impact of oxidative stress on the development of brain edema in this rat model. Methods: Oxidative stress was induced following glutathione (GSH) depletion by diethyl maleate (DEM). PCA and SHAM-operated rats received DEM (1 mg/kg/day intraperitoneally) for 10 days starting at day 18 after surgery. Rats were sacrificed at day 28 and oxidative stress markers, such as glutathione (GSH), malon-dialdehyde (MDA) and 4-hydroxy-2-nonenal (HNE) were assessed in arterial plasma and brain (frontal cortex) tissue. Plasma ammonia and liver function markers (AST, ALT, bilirubin) were also evaluated. Brain water content was measured using a specific gravimetric technique. Results: DEM induced a significant decrease in plasmatic GSH, which lead to an increase in arterial MDA (2.5 fold) and HNE (1.4 fold) levels in PCA rats compared to non-treated PCA rats. An increase in brain water content was observed in DEM-treated PCA rats vs non-treated PCA rats (PCA+DEM: 78.45 ± 0.13% vs PCA: 77.38 ± 0.11%, p< 0.001). In the brain, oxidative stress markers measured in the frontal cortex did not differ between the two groups. DEM treatment did not affect the degree of hyperammonemia or lead to an alteration in liver function in comparison to non-treated PCA rats. Conclusions: DEM induced systemic, not central, oxidative stress in PCA rats. This, imposed on hyperammonemia, resulted in an increase in brain water content. Since oxidative stress and brain edema were not detected in non-treated hyperammonemic PCA rats, together our findings suggest that a synergistic effect between hyperammonemia and systemic oxidative stress is implicated in the pathogenesis of brain edema in hepatic encephalopathy.