An effective strategy for enhancing the bioactive fatty acids (FA) in sheep milk could be dietary supplementation with a moderate level of a combination of soybean oil with fish oil (SFO) without negative effects on milk yield and its chemical composition. Thus, the objective of this study was to determine the effects of a moderate forage diet supplementation with SFO on milk chemical composition and FA profile, as well as on plasma FA. Twelve dairy sheep were assigned to two homogenous sub-groups. Treatments involved a control diet without added oil, and a diet supplemented with 23·6 g soybean oil and 4·7 g fish oil per kg dry matter (DM) of the total ration. The results showed that SFO diet had no effect on milk yield and chemical composition. In blood plasma the concentrations of trans-11 C18:2 (VA), C18:2n-6, C20:5n-3 (EPA) and C22:6n-3 (DHA) were significantly higher while those of C14:0, C16:0 and C18:0 were lower in sheep fed with SFO diet compared with control. The SFO supplementation of sheep diet increased the concentrations of VA, cis-9, trans-11 C18:2 CLA, trans-10, cis-12, C18:2 CLA, EPA, DHA, monounsaturated FA (MUFA), polyusaturated fatty acids (PUFA) and n-3 FA and decreased those of short chain FA (SCFA), medium chain FA (MCFA), the saturated/unsaturated ratio and the atherogenicity index value in milk compared with the control. In conclussion, the SFO supplementation at the above levels in a sheep diet, with moderate forage to concentrate ratio, improved the milk FA profile from human health standpoint without negative effects on its chemical composition.