This study is an exploratory analysis for understanding the effect of a duodenal infusion of an α-linolenic acid (LNA) on the plasma and milk proteome of lactating dairy cows. Four primiparous Holstein cows were fitted with duodenal cannulas and received 0, 100, 200, 300 and 400 g/day of LNA in a two-treatment crossover design. Blood and milk were collected for determination of protein composition by two-dimensional gel electrophoresis. Alteration of protein spots was detected and identified using matrix-assisted laser desorption/ionization time-of-flight/time-of-flight tandem mass spectrometry (MALDI-TOF-TOF MS). Plasma haptoglobin levels, and milk β-casein A2, αs1-casein variant and albumin, did not differ in cows after infusion of 0, 100, 200 and 300 g/day of LNA, but were increased after the cows received duodenal infusion of 400 g/day of LNA. Western blot analysis of haptoglobin expression in plasma confirmed the alterations in protein expression seen using MS. This study demonstrated that infusion of high doses of LNA by duodenal cannula can result in metabolic stress within the bovine intestine and in changes in milk composition.