Preventing obesity could be done by lowering plasma TAG that inhibits adipogenesis. Oat and mushroom beta-glucans in the diet has been reported to lower plasma lipid; however the data focusing on their effects on TAG and obesity are insufficient. In the present study, lowering plasma triacylglycerol, fat deposition, body weight gain (BWG) in rats fed a high fat diet (HFD) was evaluated. Rats in the control group were given HFD only and rats in the treatment group fed HFD enriched with 0.2%, 0.6% and 1.8% (wt:wt) beta-glucan from oats (LD-O, MD-O, HD-O) or mushroom (LD-M, MD-M, HD-M). After 6 weeks dietary intervention, the rats fed HD-M showed significantly lower plasma TAG, total fat mass, white adipose tissue, inguinal fat and BWG level more than HD-O treated rats (p < 0.05). The underlying mechanism in lowering plasma TAG, fat pad masses and BWG in HD-M was increasing ratio of fat faecal to faecal weight which was significantly higher than HD-O (p < 0.05). This study demonstrated that the preventing obesity via lowering plasma TAG and fat deposition was different depending on beta-glucan origin, either from oats and Shiitake mushroom.