Background & Aims: There are several reports suggesting hyperosmotic contents in the feces of patients suffering from inflammatory bowel disease (IBD). Previous works have documented that hyperosmolarity can cause inflammation due to methylation of the catalytic subunit of Protein Phosphatase 2A (PP2A) and subsequent Nuclear Factor-kappa B (NF-B) activation resulting in cytokine secretion. In this study, we demonstrate that Dextran Sulfate Sodium (DSS) induces colitis due to hyperosmolarity and subsequent PP2A activation. Methods: Mice were randomized and fed with increased concentrations of DSS (0 mOsm, 175 mOsm, 300 mOsm and 627 mOsm) for a duration of three weeks or, either with hyperosmotic concentrations of DSS (627 mOsm) or mannitol (450 mOsm), for a duration of twelve weeks. Results: Long-term oral administration of hyposmotic DSS or mannitol had no demonstrable effect. Hyperosmotic DSS or mannitol produced a significant increase in colonic inflammation, as well as an increase in the weight of sacral lymph nodes and in serum amyloid A protein levels. Similar results were obtained through the ingestion of comparable osmolarities of mannitol. Hyperosmolarity induces the methylation of PP2A, nuclear p65 NF-B activation and cytokine secretion. The rectal instillation of okadaic acid, a well-known PP2A inhibitor, reverses the IBD. Short inhibiting RNAs (siRNAs) targeted toward PP2Ac reverse the effect of hyperosmotic DSS. Conclusion: The present study strongly suggests that DSS-induced chronic colitis is a consequence of the methylation of PP2Ac induced by hyperosmolarity.