Aims: To determine the effects of hops extract on in vitro volatile fatty acid (VFA) production by bovine rumen micro-organisms. Methods and Results: When mixed rumen microbes were suspended in media containing carbohydrates, the initial rates of VFA production were suppressed by beta-acid-rich hops extract. The rates of VFA production increased over extended incubations (24 h), and hops extract caused an increase in the propionate to acetate ratio. Hops extract inhibited the growth and metabolism of Streptococcus bovis, but Selenomonas ruminantium and Megasphaera elsdenii were not affected. Likewise, the propionate production of M. elsdenii/S. bovis co-cultures, but not M. elsdenii/S. ruminantium co-cultures, was decreased in the presence of hops extract. Conclusions: These results are consistent with the hypothesis that the hops inhibit Gram-positive lactic acid bacteria (S. bovis), and the rumen microbial community requires a period of adaptation before normal VFA production resumes. Selenomonas bovis and S. ruminantium both produce lactate, which is the substrate for propionate production by M. elsdenii. However, S. ruminantium has an outer membrane, while S. bovis does not. Significance and Impact of Study: The enhanced production of the gluconeogenesis precursor, propionic acid, provides further evidence that plant secondary metabolites from hops could be used to improve rumen fermentation.