Obesity is a growing concern in the US and world-wide, associated with an increased risk for several cardiometabolic diseases, including metabolic associated steatotic liver disease (MASLD). Currently, therapeutic interventions to prevent and/or treat MASLD are limited, and research is needed to identify new therapeutic targets. The specific-sized 35 kDa fragment of hyaluronan (HA35), has gut protective and anti-inflammatory properties and a previous pilot clinical study reported it is well tolerated in healthy individuals. Here we tested the hypothesis that HA35 treatment ameliorates high fat diet-induced liver injury. Five-week-old male C57BL/6 J mice were allowed ad lib access to control chow or high fat fructose and cholesterol (FFC) diet over a period of 12 weeks. HA35 was administered at 15mg/kg via oral gavage on the last 6 days of the study as a therapeutic intervention. Mice on FFC diet-gained more body weight compared to those on chow diet, with final body weights ranging from 30.8 to 45.6 g. FFC diet caused hepatocyte injury, increased expression of inflammatory cytokine/chemokine mRNA, as well as indicators of liver fibrosis. When mice were stratified based on their final body weight, only mice <
40 g were protected by treatment with HA35. In this group, treatment with HA35 also restored tight junction integrity in the colon and increased expression of α -defensins in the small intestine. Taken together the data suggests that HA35 is an effective therapeutic in ameliorating high fat diet-induced liver inflammation and fibrosis in moderately obese, but not severe, conditions.