Given the human health benefits of increasing n-3 fatty acid intake, inadequate intake of these fatty acids by many people, and problems associated with supplying these fatty acids from marine sources, there is a need to increase amounts of n-3 fatty acids in foods that are frequently eaten and affordable. Therefore, this study evaluated the amount of the α-linolenic acid (ALA) that was bio-hydrogenated by bovine ruminal microbes to evaluate the potential effectiveness of a consumer-acceptable protective treatment to flaxseed. The proprietary treatment to flaxseed was evaluated with an in vitro fermentation trial that was conducted for 12 or 24 h. A small amount of quebracho tannin was included in the in vitro medium of some fermentation tubes to determine if this material could help reduce bio-hydrogenation of ALA. Residue from each tube was lyophilized then analyzed for fatty acids using direct trans-esterification with methanolic-HCl. The trial had a completely randomized design with a factorial arrangement of treatments. The treatment applied to flaxseed reduced (P < 0.01) bio-hydrogenation of ALA with 65% of ALA remaining after 24 h of fermentation versus 23% remaining for the non-treated flaxseed. Inclusion of tannin in fermentations did not help reduce bio-hydrogenation (P = 0.15), and the interaction of flaxseed treatment and tannin was not significant (P = 0.56). These results indicate that the protective treatment applied to flaxseed may reduce bio-hydrogenation of ALA in cattle and this may lead to greater amounts of n-3 fatty acids in their muscles if they consume the treated flaxseed daily for several months before harvest.