Abstract The objective of this study was to examine the effect of ruminal pulse dose of free linoleic acid (LA) and free docosahexaenoic acid (DHA) on microbial populations in the rumen, duodenal fatty acid (FA) flow, milk composition, and milk FA profiles of Chinese Holstein dairy cows. Four rumen- and duodenal-fistulated Chinese Holstein cows in mid lactation (138.5±10d in milk) were randomly assigned to 3 treatment groups and 1 control group in a 4×4 Latin square design over 4 periods (3 wk per period). Diets contained either no LA or 2.7% LA and either no DHA or 0.5% DHA in a 2×2 factorial arrangement of treatments. Ruminal pulse dose with DHA increased counts of Megasphaera elsdenii, decreased Fibrobacter succinogenes, but did not affect Butyrivibrio fibrisolvens or Ruminococcus flavefaciens. The pulse dose of LA at 2.7% dry matter had no effect on the population sizes of the 3 major cellulolytic bacterial species or M. elsdenii, and no interaction was observed between LA and DHA. The pulse dose of LA or DHA, either alone or in combination, increased the duodenal flow of vaccenic acid (VA). The milk VA and cis-9,trans-11 conjugated linoleic acid (CLA) contents also increased in response to the fatty acid pulse dose, and the pulse dose of both LA and DHA together had the most profound stimulatory effect. This study indicated that ruminal pulse dose of LA or DHA could be used to increase duodenal flow of VA and the milk contents of potentially health-promoting FA, such as VA and cis-9,trans-11 CLA. These results might be useful in formulating dietary interventions to improve milk cis-9,trans-11 CLA contents.