As a dietary supplement, hyaluronic acid (HA) has exhibited appreciable immunomodulatory activity and an ameliorative effect on rodent colitis. However, its high viscosity is not only refractory to absorb through the gut, but also causes flatulence. In contrast to HA, hyaluronic acid oligosaccharides (o-HAs) can overcome the above-mentioned constraints, but their treatment effect still remains ill-defined contemporarily. Herein, the current study intends to compare the modulatory effects of HA and o-HA on colitis and assess the underlying molecular mechanism. We first showed that o-HA had a better preventive effect than HA in alleviating colitis symptoms, as evidenced by lower body weight loss, lower disease activity index scores, a lower inflammatory response (TNF-α, IL-6, IL-1β, p-NF-κB), and more intact colon epithelial integrity in vivo. The best efficiency was observed in the o-HA treated group with a dosage of 30 mg kg-1. In an in vitro barrier function assay, o-HA exerted a better protective effect on the transepithelial electrical resistance (TEER), FITC permeability, and wound healing and modulated the expression of tight junction (TJ) proteins (ZO-1, occludin) in lipopolysaccharide (LPS)-stimulated Caco-2 cells. In summary, both HA and o-HA showed the potential to reduce inflammation and ameliorate intestinal damage in DSS-induced colitis and LPS-induced inflammation, but o-HA had improved outcomes. The results also provided a glimpse of the latent mechanism by which HA and o-HA enhanced intestinal barrier function via MLCK/p-MLC signaling pathway suppression.