AimsThis study was conducted to isolate and identify propionate-producing bacteria that can be used as an inoculum in improving wet brewers grains and rumen fermentation via increasing propionate concentration. Methods and ResultsA strain of Lactobacillus that exhibits high levels of propionate production was identified and characterized as Lactobacillus mucosae 521129 by 16S rRNA gene sequencing and phylogenetic analyses. Wet brewers grains were fermented through L. mucosae inoculation and resulted in an increase in propionate concentration. Fermented wet brewers grains were used in invitro rumen fermentation and revealed that L. mucosae-fermented wet brewers grains produced more gas and had higher accumulations propionate and total volatile fatty acid (VFA) than the control. The fewest methanogen DNA copies were detected in L. mucosae-fermented wet brewers grains. ConclusionIdentified L. mucosae improved the fermentation of wet brewers grains and the invitro rumen fermentation via increasing propionate and total VFA concentrations. Significance and Impact of the StudyThe presented research provided the identification of L. mucosae 521129 as a propionate producer and was metabolically profiled. Furthermore, data present the putative application of this organism in improving the fermentation of wet brewers grains and invitro rumen fermentation.