The n-6/n-3 metabolic pathway associated with hepatic glycerolipid portioning plays a key role in preventing obesity. In this nutrition metabolism study, we used in vivo monitoring techniques with 40 obese male Sprague-Dawley strain rats attached with jugular-vein cannula after obesity was induced by a high-fat diet to determine the molecular mechanism associated with hepatic glycerolipid partitioning involving the n-6/n-3 metabolic pathway. Rats were randomly assigned to four groups (10 animals per group), including one control group (CON, n-6/n-3 of 71:1) and three treatment groups (n-6/n-3 of 4:1, 15:1 and 30:1). They were fed with experimental diets for 60 days. Incorporation rates of [14C]-labeling lipid into glycerolipid in the liver were 28.87-37.03% in treatment groups fed with diets containing an n-6/n-3 ratio of 4:1, 15:1 and 30:1, which were significantly (p < 0.05) lower than that in the CON (40.01%). However, 14CO2 emission % of absorbed dose showed the opposite trend. It was significantly (p < 0.05) higher in a treatment groups (n-6/n-3 of 4:1, 15:1 and 30:1, 30.35-45.08%) than in CON (27.71%). Regarding the metabolic distribution of glycerolipid to blood from livers, phospholipid/total glycerolipid (%) was significantly (p < 0.05) lower in CON at 11.04% than in treatment groups at 18.15% to 25.15%. Moreover, 14CO2/[14C]-total glycerolipid (%) was significantly (p < 0.05) higher in treatment groups at 44.16-78.50% than in CON at 39.50%. Metabolic distribution of fatty acyl moieties flux for oxidation and glycerolipid synthesis in the liver were significantly (p < 0.05) better in order of 4:1 > 15:1 > 30:1 than in the CON. Our data demonstrate that n-6/n-3 of 4:1 could help prevent obesity by controlling the mechanism of hepatic partitioning through oxidation and esterification of glycerolipid in an obese animal biomodel.