The aim of this study was to evaluate the performance of cows fed with a total mixed ration composed of elephant grass and a concentrate containing 0.0% (control), 1.5%, 3.0% or 4.5% soybean oil on a dry matter basis. The effect of milking time (morning versus afternoon) on butter fatty acid composition was also evaluated. Twelve multiparous Holstein x Gyr cows with an average milk production of 18.0±4.6 kg day-1 and 90±25 days in milk were used in a triplicated 4 x 4 Latin square experimental design with 15-day periods. Data were analyzed using mixed models. There was no effect of soybean oil supplementation on dry matter or neutral detergent fiber intake (P > 0.05). The fat-corrected milk yield linearly decreased (P=0.0109) and the milk protein yield linearly increased (P=0.0023) in response to soybean oil supplementation. The butterfat trans-9 C18:1, cis-9 C18:1, trans-10 C18:1, trans-11 C18:1, cis-9, trans-11 CLA and trans-10, cis-12 CLA content linearly increased (P < 0.05), whereas the C12:0, C14:0 and C16:0 content linearly decreased (P < 0.001) as the level of soybean oil in the diet increased. The butter produced from afternoon milk had a lower content of C16:0 and a higher content of cis-9 C18:1 (P < 0.05). The supplementation of elephant grass-based-diets with soybean oil and, to a small extent, the selective segregation of milk obtained from afternoon milking sessions are strategies that can be used to improve the fatty acid composition of butterfat.