The protective effect of creatine supplementation on the jejunal epithelium of rats under conditions of oxidative stress induced by chronic acidosis was investigated. In particular, we measured the activities of the main antioxidant enzymes (superoxide dismutase, glutathione peroxidase, catalase, glutathione reductase), the level of lipid peroxidation, the expression of heat shock proteins (HSP70), and the expression of the major carriers of the cells (Na+/K+-ATPase, SGLT1 and GLUT2) in control and chronic acidosis conditions. Creatine did not affect the activity of antioxidant enzymes, nor in control, neither in acidosis, except for catalase, which activity was reduced in both conditions. Creatine did not change the level of lipid peroxidation and the expression of HSP70. Finally, creatine stimulated the expression of (Na+/K+)-ATPase both in control and in chronic acidosis. Chronic acidosis caused a reduction in the expression of GLUT2 and SGLT1. GLUT2 reduction was abolished by creatine, while the presence of creatine did not induce any strengthening effect on the expression of SGLT1, neither in control nor in chronic acidosis. These results indicate that creatine has antioxidant properties that would be realized through direct interaction of the molecule with reactive oxygen species. Moreover, the administration of creatine seems to determine a functional strengthening of the tissue making the tissue more resistant to acidosis.