The effects of ruminant diet supplementation with linoleic or different polyunsaturated fatty acids (FA) have been well documented. Less abundant information, however, exists on the effects of incorporating monounsaturated FA, such as oleic acid, on lipid metabolism or animal performance. The purpose of this work was to assess the effects of feeding dairy ewes a diet supplemented with high levels of olive oil (OO) on milk yield and composition, paying particular attention to the FA profile. Twenty-four Assaf ewes were fed ad libitum with 2 diets, control or supplemented with 6% OO (2 lots of 6 animals per diet) for 4 wk. Milk yield and composition and dry matter intake were recorded weekly. Milk FA composition was determined by gas chromatography and conjugated linoleic acid profile by silver ion HPLC. Milk yield increased in ewes receiving OO, with no differences in dry matter intake. The OO diet decreased the milk protein percentage but increased the milk fat, protein, and total solids yield. Medium-chain saturated FA (C10:0 to C16:0) content was reduced with OO supplementation, whereas C18:0 and cis-9 C18:1 content increased. Leaving aside trans-11, most trans C18:1 isomers, mainly trans-10, increased in supplemented ewes. The main conjugated linoleic acid isomer (cis-9, trans-11 C18:2) decreased with OO supplementation, whereas trans-7, cis-9 and trans-9, cis-11 C18:2 exhibited a remarkable increase. These results support the argument that the supplementation of ewe diets with high levels of OO does not have any detrimental effects on animal performance but substantially modifies the FA profile.