This study evaluated the effect of fish oil emulsion supplementation (rich in ω-3 fatty acids) on synaptosomal Na+,K+-ATPase and K+-dependent-paranitrophenyl phosphatase (K+-pNPPase) activities in diabetic rats. Diabetes mellitus was induced in 36 rats by injection of streptozotocin (STZ) via the tail vein at a dose of 45mg/kg of body weight. After 7 days, animals were randomly divided into six groups according to the administration and doses of the fish oil emulsion: (1) control rats, (2) untreated diabetic rats, (3) control rats treated with the 45% fish oil emulsion at a dose of 160mg/kg/day, (4) control rats treated with a dose of 800mg/kg/day, (5) diabetic rats treated with the fish oil emulsion at a dose of 160mg/kg/day, and (6) diabetic rats treated with a dose of 800mg/kg/day. The composition of fish oil emulsion was: 8.1% eicosapenthaenoic acid (EPA, 20:5, ω-3), 5.9% DHA (22:6, ω-3). We found a non-significant 26% reduction of Na+,K+-ATPase activity (0.724nkat/mg vs. 0.974nkat/mg proteins) and no change in K+-pNPPase activity in diabetic synaptosomes vs. control group. 7-wk fish oil supplementation indicated a non-significant restoration of Na+,K+-ATPase activity in diabetic synaptosomes compared to untreated diabetic group. In addition, a significantly protective effect of supplementation with the fish oil emulsion on the K+-pNPPase activity only in non-diabetic animals was observed. However, there was no effect of the fish oil emulsion supplementation on the enzyme activity in diabetic animals. The findings with different effects of experimental diabetes and the fish oil emulsion supplementation on Na+,K+-ATPase and K+-pNPPase activities may support the possibility of additional function of K+-pNPPase outside the ATPase complex. Another possibility is that ROS or products of oxidative damage to macromolecules might influence these two enzyme activities by a different mechanism.