Four ruminally cannulated Holstein cows averaging 43 days in milk (DIM) were used in a $4{\times}4$ Latin square to determine the effect of feeding extruded soybean, ground canola seed and whole cottonseed on ruminal fermentation and milk fatty acid profile. One hundred and twenty lactating Holstein cows, 58 (${\pm}31$) DIM, were assigned to four treatments in a completely randomized block design to study the effects of the three types of oilseeds on production parameters and milk fatty acid profile. The four diets were a control diet (CON) and three diets in which 10% extruded soybean (ESB), 5% ground canola seed (GCS) and 10% whole cottonseed (WCS) were included, respectively. Diets consisted of concentrate mix, corn silage and Chinese wild rye and were balanced to similar concentrations of CP, NDF and ADF. Ruminal fermentation results showed that ruminal fermentation parameters, dry matter intake and milk yield were not significantly affected by treatments. However, compared with the control, feeding cows with the three oilseed diets reduced C14:0 and C16:0 and elevated C18:0 and C18:1 concentrations in milk, and feeding ESB increased C18:2 and cis9, trans11 conjugated linoleic acid (CLA). Production results showed that feeding ESB tended to increase actual milk yield (30.85 kg/d vs. 29.29 kg/d) and significantly decreased milk fat percentage (3.53% vs. 4.06%) compared with CON. Milk protein (3.41%) and solid non-fat (13.27%) from cows fed WCS were significantly higher than from cows fed CON (3.24% and 12.63%, respectively). Milk urea N concentrations from cows fed the ESB (164.12 mg/L) and GCS (169.91 mg/L) were higher than cows fed CON (132.31 mg/L). However, intake of DM, 4% fat corrected milk, energy corrected milk, milk fat and protein yields, milk lactose percentage and yield, somatic cell count and body condition score were not affected by different treatments. The proportion of medium-chain fatty acid with 14 to 16 C units in milk was greatly decreased in cows fed ESB, GCS and WCS. Feeding ESB increased the concentration in milk of C18:1, C18:2, C18:3 and cis9, trans11-CLA content by 16.67%, 37.36%, 95.24%, 72.22%, respectively, feeding GCS improved C18:0 and C18:1 by 17.41% and 33.28%, respectively, and feeding WCS increased C18:0 by 31.01% compared with feeding CON. Both ruminal fermentation and production trial results indicated that supplementation of extruded soybean, ground canola seed and whole cottonseed could elevate the desirable poly- and monounsaturated fatty acid and decrease the medium chain fatty acid and saturated fatty acid content of milk fat without negative effects on ruminal fermentation and lactation performance.