Aims: To evaluate the effects of treating barley grain with lactic acid (LA) and heat on postprandial dynamics of 19 microbial taxa and fermentation in the rumen of dairy cows. Methods and Results: This study was designed as a double 3 9 3 Latin square with six rumen-cannulated cows and three diets either containing untreated control barley or barley treated with 1% LA and 1% LA and heat (LAH, 55 degrees C). Microbial populations, pH and volatile fatty acids were assessed in rumen liquid and solids during the postprandial period. Propionate increased and butyrate decreased in rumen solids of cows fed LA and LAH treated barley compared to the control barley. The LA but not LAH treatment depressed Fibrobacter succinogenes in rumen liquid and solids, whereas the opposite effect was observed for Ruminococcus albus in both fractions and Ruminococcus flavefaciens in rumen solids. LA promoted Ruminobacter amylophilus with the effect being more pronounced with LAH. The Lactobacillus group and Megasphaera elsdenii increased in both fractions with LA but not with LAH. Conclusions: LA and LAH treatment of barley differently altered ruminal abundance of certain bacterial taxa and fungi and increased propionate fermentation in rumen solids, whereby LA and LAH effects were consistent and mostly independent of the rumen fraction and time after barley feeding. Significance and Impact of the Study: Results provided evidence that LA and LAH treatment of barley can enhance rumen propionate fermentation without adversely affecting rumen pH. As propionate is the major contributor to gluconeogenesis in ruminants, the present barley treatment may have practical application to enhance energy supply in dairy cows.