Ulcerative colitis (UC) is one of the inflammatory bowel diseases characterized by colonic damage. Epigenetic mechanisms are suggested to play a role in the pathogenesis of UC. Pioglitazone has shown promise for the treatment of UC; however, the role of epigenetic pathways in this effect is unclear. The current study aimed to explore the therapeutic and protective effects of pioglitazone against acetic acid-induced colitis (AA-C) in rats and the role of epigenetic modulation and antioxidant mechanisms in this effect. Forty male albino rats were divided into four groups (n = 10/group): control (normal saline), acetic-acid-induced ulcerative colitis (AA-C) (3 days, 2 ml acetic acid 4%), pioglitazone-treated (AA, followed by 3-week oral pioglitazone 25 mg/kg/day), and pioglitazone-protected groups (3-day oral pioglitazone 25 mg/kg/day before AA, continued with AA, and 3 weeks later). After the experiment, the body weight, colon weight-to-length ratio, and colonic tissue were evaluated. The colonic expression of epigenetic markers (DNA methyltransferase- 1 and methylated E-cadherin), oxidative stress marker (malondialdehyde), antioxidant enzyme (superoxide dismutase), and angiotensin-converting enzyme- 2 (ACE- 2) was evaluated. The pioglitazone-protected and treated groups showed significant inhibition of DNA methyltransferase- 1 and methylated E-cadherin with improvement in colonic tissue macroscopic and microscopic signs of inflammation, improved weight, less oxidative stress, and less ACE- 2 expression. These beneficial actions were more pronounced among the pioglitazone-protected group. Pioglitazone could mitigate AA-C in rats by inhibiting epigenetic DNA methyltransferase- 1 and E-cadherin gene methylation. It also inhibits oxidative stress and prevents the overexpression of ACE- 2.