During an in vivo trial with four rumen-fistulated dairy cows, the effect of dietary supplementation of docosahexaenoic acid (DHA; 22:6 n-3)-enriched microalgae (Schizochytrium sp., 2% on feed intake) on the fatty acid metabolism and microbial community in the rumen was investigated. Algal supplementation resulted in lower concentrations of saturated fatty acids while the concentrations of monounsaturated and polyunsaturated fatty acids increased as compared to the control treatment. An incomplete biohydrogenation of linoleic (18:2 n-6) and linolenic (18:3 n-3) acid was observed, resulting in a strong accumulation of 18:1 t11, 18:1t10 and 18:2 t11c15. Denaturing gradient gel electrophoresis (DGGE) was performed to study the rumen bacterial and ciliate population. Algal supplementation provoked changes in both communities. Ciliate DGGE profiles suggested a decreased abundance of Isotricha prostoma and I. intestinalis and some species of Epidinium caudatum, Eudiplodinium maggii and Diplodinium dentaturn in the rumen of algae-fed cows. Moreover, quantitative PCR indicated that incomplete biohydrogenation through algal addition was associated with decreased ciliate numbers. The simultaneous accumulation of hydrogenation intermediates and the disappearance of ciliates suggest that ciliates and/or their associated bacteria could play a role in rumen biohydrogenation.