Enteric methane constitutes an energy loss for the animal and contributes to global warming and climate change. Stearidonic acid (SDA; C18:4n-3), a highly polyunsaturated n-3 fatty acid, could have potential to reduce methane production. Effects of increasing levels of SDA on methane production were evaluated in short-term batch incubations (24 h) of a mixed diet with buffered rumen fluid. Stearidonic acid was supplemented at 0 (SDA0; control), 1 (SDA1), 5 (SDA5), 20 (SDA20), and 50 (SDA50) mg/L incubation media. Increasing levels of SDA supplementation had no effect on total gas (mL) and methane production (mmol/g TMR dry matter, DM), or total concentration of volatile fatty acids (VFA; mmol/L). Stearidonic acid induced a shift to increased propionate production at the expense of acetate and butyrate, with the largest effect at the highest inclusion level. The apparent biohydrogenation of stearidonic acid was extensive, with less than 2% being detected after 24 h of incubation, and only at the highest level of addition. Increasing levels of SDA promoted an accumulation of C18:2 and C18:1 isomers, particularly vaccenic acid (trans-11 C18:11), with no effect on the end-product (stearic acid; C18:0). Effects on fermentation pattern and SDA biohydrogenation were not associated with a reduction in methanogenesis, suggesting that either higher levels of non-esterified SDA or supplementation in an alternative form might be needed to achieve methane mitigation.