Four Holstein cows were used in a 4 × 4 Latin square design to determine the effect of feeding different processing whole cottonseed (WCS) on milk yield, milk composition and milk fatty acid profi le. Result showed that dry matter intake and milk yield were unaffected by different treatments, but milk protein concentrations were increased in cows fed processing WCS diets. Compared with control, feeding cows with different processing WCS diets reduced C14:0 and C16:0 and elevated C18:0 and C18:1 in milk, and feeding extruded delinted WCS increased C18:2 and cis9, trans11 conjugated linoleic acid in milk.