An increased consumption of n-3 fatty acids (omega-3 FA) has been associated with various health benefits. Altering the fatty acid composition of milk fat is a desirable means to increase human n-3 FA consumption. The aim of this study was to investigate the effect of dietary supplementation with a docosahexaenoic acid (DHA) rich microalgae on rumen fermentation and milk fatty acid profile. The trial was conducted using 2 rumen-fistulated dairy cows, fed for a 2-week pre-trial feeding period (Control, CON), 4 weeks on the treatment diet (CON supplemented with 140g/cow/day of Aurantiochytrium limacinum; CCAP 4087/2, ALL-G-RICH, Alltech Inc.), followed by a 1-wk washout period (CON). Rumen fluid samples were taken 4 times per week providing 28 samples per animal over the course of the experiment. Rumen fluid samples were taken before milking and analyzed by GC for short chain fatty acids (SCFA), and bacterial yield and specific microbial populations (lactobacilli, methanogens, Veillonella spp., Selenomonas ruminantum, Fibrobacter succinogenes, Megasphaera elsdenii and Clostridal Cluster XIVa) were measured by quantitative RT-PCR using appropriate primers. Milk samples (n = 28/animal) were taken on the same days as the rumen fluid samples. The addition of microalgae had no effect on SCFA or the bacterial density in the rumen fluid. Milk fat concentration was unaffected but DHA content increased 100-fold during the 4 weeks of supplementation. Numbers of lactobacilli and methane-producing bacteria were reduced, while the number of beneficial fiber-degrading bacteria increased with microalgae addition to the diet. These results indicate that the addition of microalgae under these conditions does not depress rumen fermentation or milk fat content but significantly increases milk DHA. The effect of microalgae supplementation on Fibrobacter succinogenes merits further investigation.