Background: Arginine has been shown to have several immunological and trophic properties in stressful diseases. Its metabolites, nitric oxide (NO) and polyamines, are related to arginine’s effects. Thus, the aim of this study was to determine the effects of the NO donor L-arginine and the role of inducible NO synthase (iNOS) on intestinal permeability and bacterial translocation in a model of intestinal obstruction (IO) induced by a simple knot in the terminal ileum. Material and Methods: Male C57BL6/J wild-type (WT) and iNOS knockout (iNOS–/–) mice were randomized into 6 groups: Sham and Sham–/– (standard chow), IO and IO–/– (standard chow +IO), and Arg and Arg–/– (standard chow supplemented with arginine + IO). After 7 days of treatment with standard or supplemented chows, IO was induced and intestinal permeability and bacterial translocation were evaluated. The small intestine and its contents were harvested for histopathological and morphometric analysis and the determination of polyamine concentration. Results: Pretreatment with arginine maintained intestinal permeability (P > .05; Arg and Arg–/– groups vs Sham and Sham–/– groups), increased polyamine concentration in intestinal content (P < .05; Arg vs IO group), and decreased bacterial translocation in WT animals (Arg group vs IO and IO–/–groups). Absence of iNOS also presented a protective effect on permeability but not on bacterial translocation. Conclusion: Arginine supplementation and synthesis of NO by iNOS are important factors in decreasing bacterial translocation. However, when intestinal permeability was considered, NO had a detrimental role.