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Plant sterols (PSs) have reported benefits in alleviating colitis in mice, but the mechanisms involved require further investigation. This study aimed to evaluate the effect of a dietary supplement containing PSs (PS-DS) on inflammation biomarkers in a mice model of chronic ulcerative colitis induced by dextran sulfate sodium (DSS). C57BL/6J mice (n = 34) were exposed to 1.5% DSS in drinking water for three 5-day periods, with 10-day rest intervals in between. The mice received daily PS-DS (35 mg PS/kg) or placebo by oral gavage, either simultaneously (treatment) or 30 days prior (pre-treatment) to DSS exposition. After euthanasia, myeloperoxidase (MPO) activity and the levels of pro- (TNF-α and IL-6) and anti-inflammatory cytokines (IL-10) in the colonic tissue were analyzed. PS-DS treatment reduced (vs. DSS + placebo) myeloperoxidase (MPO) activity (2.6 ± 0.2 vs. 2.1 ± 0.1-fold change) and levels of TNF-α (85 ± 11 vs. 39 ± 7 pg/mg protein) and IL-6 (214 ± 26 vs. 128 ± 18 pg/mg protein), increasing the levels of IL-10 (46 ± 5 vs. 136 ± 16 pg/mg protein). PS-DS pre-treatment provided a greater inhibition (vs. treatment) of MPO activity (2.1 ± 0.3 vs. 1.3 ± 0.1-fold change) and a greater increase in IL-10 levels (50 ± 9 vs. 178 ± 30 pg/mg protein). These findings suggest that PS-DS has the potential to alleviate colitis in mice by modulating the inflammatory response and reducing oxidative stress. However, studies in humans are required to validate and fully understand its anti-inflammatory effect. [ABSTRACT FROM AUTHOR]