Six midlactation Holstein cows were fed a total mixed ration supplemented with either 4.8% canola meal, 3.3% unprotected canola seeds plus 1.5% canola meal, or 4.8% formaldehyde-protected canola seeds, according to a double 3 x 3 Latin square design. Each period lasted 3 wk; experimental analyses were restricted to the last week of each period. Mammary biopsies were taken the last day of each period for gene expression measurements. Milk production and milk protein percentage were reduced by canola seeds, whether protected or unprotected. Protected canola seeds also decreased dry matter intake. Feeding canola seeds reduced the content of C-8 to C-16 fatty acids in milk and increased the content of oleic acid (C-18:1c9) Unprotected canola seeds elevated the concentrations of C-18:0 Protected canola seeds increased the C-18:2 and C-18:3 content, and reduced the C-18:0/C-18:1c9 ratio. Similar results were obtained for plasma fatty acids, with some specific features, such as an increased C-16:0/C-16:1 ratio with protected canola seeds. Canola seeds had no significant effects on insulin, triglycerides, or cholesterol present in serum, but increased the concentration of nonesterified fatty acids; a greater increase was obtained with protected canola seeds. Expression levels of acetyl-CoA carboxylase and delta 9-stearoyl-CoA desaturase genes measured in the mammary gland did not differ significantly between diets. Therefore, the reduced C-18:0/C-18:1c9 ratio observed in milk with protected canola seeds was not due to an enhanced expression of the delta-9 desaturase in the mammary gland.