Hyperglycemia and insulin resistance are the common metabolic disorders in type 2 diabetes mellitus.  This study was designed to evaluate the anti-diabetic effect of fish oil from a hybrid catfish (Pangasianodon hypophthalmus x Pangasius bocourti) on high-fat diet and streptozotocin (STZ) induced type 2 diabetes rats.  Male Wistar rats were divided into five groups: normal rats as control (NC), normal rats receiving fish oil (NFO), type 2 diabetic rats as control (DMC), type 2 diabetic rats receiving fish oil at a dose of 1000 mg/kg/kg BW/day (DMFO) and type2 diabetic rats treated with metformin (DMD) at 50 mg/kg BW/day throughout the experimental period as a positive drug.  At the end of the study, 12 weeks of supplement with fish oil successfully decreased plasma glucose level and also ameliorated the insulin resistance in the DMFO group when compared to the DMC group.  Corresponding, the insulin-stimulated glucose uptake assessed in vitro study by the isolated hemi-diaphragm was significantly increased in the DMFO group while the basal glucose uptake did not affect via fish oil supplement in diabetic rats.  According to these present findings, the supplement of fish oil from a hybrid catfish (Pangasianodon hypophthalmus x Pangasius bocourti) possessed an anti-diabetic effect on the type 2 diabetic rat model. It exerted an antihyperglycemic effect partly via an enhancing of insulin-stimulated muscle glucose uptake and thereby improving insulin sensitivity.