Oil seeds are natural sources of fat and protein in diets for lactating cows, and are usually fed whole or crushed. A recently released variety of safflower seed, 'Nutrasaff,' contains high fat (47% crude fat) and low fiber (26% NDF), and has a potential to be effectively used as a fat supplement for lactating dairy cows. Therefore, a lactating dairy cow trial was conducted to assess production performance of dairy cows when fed graded levels of whole Nutrasaff safflower seed (NSS), to determine the optimum level of NSS supplementation in the diet and to identify its impact on milk fat content and milk fatty acid (FA) profiles. Fifteen Holstein dairy cows in midlactation (118 ± 39 days in milk) were assigned into 5 groups of 3 cows each according to previous milk yield. The experimental design was a triple 5 × 5 Latin square with each period lasting 21 d (14 d of treatment adaptation and 7 d of data collection). The animals were fed a basal diet containing 56% forage (69% alfalfa hay and 31% corn silage) and 44% concentrate mix. The diet was supplemented with 0 (control), 1, 2, 3, or 4% (DM basis) whole NSS. The NSS was added to the diet by replacing whole linted-cottonseed. Intake of DM ranged from 26.4 to 27.5 kg/d across all treatments, and did not differ due to NSS inclusion. Yield of milk and ECM averaged 33.7 and 31.6 kg/d, respectively, and they were similar in response to NSS inclusion. Milk fat percentage decreased with increasing NSS inclusion, while milk protein and lactose concentrations did not differ among treatment diets. Milk fat concentration was reduced by 11% when NSS was included at 4% of the dietary DM. Feeding NSS at 1, 2, or 3% resulted in a similar milk fat concentration, and these diets also had similar milk fat percentage compared with the control diet. Concentration of milk urea N decreased by NSS inclusion regardless of level of NSS inclusion, implying that NSS supplementation improved dietary N use for milk production. Digestibilities of DM (P = 0.12) tended to increase when NSS was supplemented at 1, 2, or 3%. Cis-9, trans-11 conjugated linoleic acid (CLA) linearly increased as the NSS inclusion increased. Total concentration of n-3 FA increased by feeding NSS at 1 and 2%, whereas total concentration of n-6 FA linearly increased with increasing inclusion level of NSS. This study clearly demonstrates that it is highly possible to use NSS as a means of fat supplementation to lactating dairy cows without negative impact on lactational performance if added less than 3% of dietary DM. The enhanced milk quality with increased cis-9, trans-11 CLA concentration due to the addition of NSS could have positive implications to human health.