Feeding unsaturated oils to lactating dairy cows impair ruminal biohydrogenation (BH) of unsaturated fatty acids (USFA) and increase ruminal outflow of BH intermediates such as trans-10, cis-12 CLA that are considered to be potent inhibitors of milk fat synthesis. Supplementing lactating dairy cow's rations containing plant origin oils with monensin and/or vitamin E may minimise the formation of trans-10 isomers in the rumen, thereby preventing milk fat depression. Therefore, this study was conducted to evaluate the effects of monensin and vitamin E supplementation in the diets of lactating dairy cows containing whole cottonseed, as the main source of FA on feed intake, milk production and composition, milk fatty acid profile, efficiency of nitrogen (N) utilisation, efficiency of net energy (NE) utilisation and nutrients digestibilities. Four multiparous Holstein lactating dairy cows (86±41 days in milk) were assigned to a balanced 4 × 4 Latin square design. Each experimental period lasted 21 days with a 14 days of treatment adaptation and a 7 days of data collection. The control diet was a total mixed ration (TMR) consisted of 430 g/kg forage and 570 g/kg of a concentrate mixture on dry matter (DM) basis. Cows were randomly assigned to one of the four dietary treatments including control diet (C), control diet supplemented with 150 mg of vitamin E/kg of DM (E), control diet supplemented with 24 mg of monensin/kg of DM (M) and control diet supplemented with 150 mg of vitamin E and 24 mg of monensin/kg of DM (EM). Dry matter intake (DMI) ranged from 19.1 to 19.5 kg/d and was similar among the dietary treatments. Dietary supplementation with vitamin E or monensin had no effect on milk production, milk fat, protein and lactose concentrations, efficiency of utilisation of nitrogen and net energy for lactation (NEL ). Digestibility of DM, organic matter (OM), crude protein (CP) and ether extract (EE) was not affected by the dietary treatments. Digestibility of neutral detergent fibre (NDF) was higher in cows fed with the M and EM diets in relation to those fed the C and E diets. The concentrations of C4:0, C6:0, C8:0, C10:0, C12:0, C14:0, C15:0, trans-10-16:1, cis-9-16:1, 17:0, 18:0, trans-11-18:1, cis-9-18:1, cis-9, trans-11 conjugated linoleic acid (CLA), trans-10, cis-12 CLA, and 18:3n-3 FA in milk fat were not affected by the dietary supplementations. While feeding the M diet tended to decrease milk fat concentration of C16:0, the milk fat concentration of C18:2n-6 FA tended to be increased. Dietary supplementation with vitamin E or monensin had no effect on milk fat concentrations of saturated, unsaturated, monounsaturated, polyunsaturated, short chain and long chain FA, but feeding the M diet numerically decreased milk fat concentration of medium chain fatty acids (MCFA). The results showed that vitamin E and/or monensin supplementations did not improve milk fat content and did not minimise the formation of trans-10 FA isomers in the rumen when whole cottonseed was included in the diet as the main source of fatty acids.