Globally, the incidence and prevalence rates of ulcerative colitis (UC) show a rising pattern. The limited efficacy and significant adverse effects associated with current treatment options underscore the need for novel therapeutic approaches. It has been found that linagliptin, a dipeptidyl peptidase-4 inhibitor, activates AMPK in different disease conditions. The main objective of the present work was to elucidate the potential implications of the AMPK/FOXO3a mediated by linagliptin in rats with chronic colitis. The findings of the current report revealed the first robust in-vivo evidence advocating the coloprotective effect of linagliptin against dextran sodium sulfate-induced chronic UC in rats. It has demonstrated potential beyond its antidiabetic effects by modulating FOXO3a localization. By shifting FOXO3a from the cytosol to the nucleus, linagliptin enhanced the transcription of genes involved in attenuation of pro-inflammatory events and restoration of redox homeostasis. Nuclear FOXO3a also impacted NFkappaB activity, reducing inflammation. This conclusion was fundamentally supported by the documented improvements in histopathological changes evidenced by reduced inflammation, edema, crypt atrophy, and submucosal fibrosis. Moreover, decreased colon weight/length ratio, as well as reduced scores of disease activity and macroscopic damage indices, were observed. Furthermore, it corrected body weight loss during the time frame of the experiment. These findings underscore the anti-inflammatory potential of therapies that promote the nuclear localization of FOXO3a in inflammatory conditions. Linagliptin's ability to modulate FOXO3a localization might be particularly useful for diabetic patients suffering from inflammatory bowel diseases. However, further molecular investigations are required to validate the findings and to assess the clinical application of this approach as a valid tool for alleviating UC.