The potential of a plant sterol food supplement (PS-FS) in addressing intestinal inflammation in vivo and in vitro was evaluated. As in vivo model, C57BL/6J mice were exposed to 1.5% dextran sulfate sodium in three 5-day periods, with 10-day rest intervals in between, daily reciving PS-FS (35 mg PS/kg body weight). The in vitro approach involved a bi-cameral system with 8-day-differentiated Caco-2 (apical) and RAW264.7 cells (basolateral). The bioaccessible fraction of PS-FS, obtained after INFOGEST 2.0 simulated gastrointestinal digestion, was added to the apical part (90 min), followed by stimulation with lipopolysaccharide (1 µg/mL, 24 h). PS-FS alleviated rectal bleeding and rebalanced pro- (tumor necrosis factor α, interleukin (IL)-6, and IL-8) and anti-inflammatory cytokines (IL-10). Additionally, PS-FS ameliorated histopathological damage and enhancing occludin expression. A reduction in oxidative stress was evidenced by decreased myeloperoxidase activity and reactive oxygen species production. The anti-inflammatory mechanism included suppressing cyclooxygenase 2 expression, reducing prostaglandin E₂ production, and inhibiting the phosphorylation and nuclear translocation of the nuclear factor κB p65 subunit. This study reveals the potential of PS-FS as a therapeutic intervention for colitis. The alignment between in vivo and in vitro outcomes substantiates the appropriateness of the co-culture model to evaluate the anti-inflammatory activity of foods.