Background:Endolysins, the bacteriophage-originated peptidoglycan hydrolases, are a promising replacement for antibiotics due to immediate lytic activity and no antibiotic resistance. The objectives of this study were to investigate the lytic activity of endolysin LyJH307 against S. bovis and to explore changes in rumen fermentation and microbiota in an in vitro system. Two treatments were used:1) control, corn grain without LyJH307;and 2) LyJH307, corn grain with LyJH307 (4 U/mL). An in vitro fermentation experiment was performed using mixture of rumen fluid collected from two cannulated Holstein steers (450 ± 30 kg) and artificial saliva buffer mixed as 1:3 ratio for 12 h incubation time. In vitro dry matter digestibility, pH, volatile fatty acids, and lactate concentration were estimated at 12 h, and the gas production was measured at 6, 9, and 12 h. The rumen bacterial community was analyzed using 16S rRNA amplicon sequencing. Results:LyJH307 supplementation at 6 h incubation markedly decreased the absolute abundance of S. bovis (approximately 70％compared to control, P=0.0289) and increased ruminal pH (P=0.0335) at the 12 h incubation. The acetate proportion (P=0.0362) was significantly increased after LyJH307 addition, whereas propionate (P= 0.0379) was decreased. LyJH307 supplementation increased D-lactate (P=0.0340) without any change in L-lactate concentration (P>0.10). There were no significant differences in Shannon\'s index, Simpson\'s index, Chao1 estimates, and evenness (P>0.10). Based on Bray-Curtis dissimilarity matrices, the LyJH307 affected the overall shift in microbiota (P=0.097). LyJH307 supplementation induced an increase of 11 genera containing Lachnoclostridium, WCHB1–41, unclassified genus Selenomonadaceae, Paraprevotella, vadinBE97, Ruminococcus gauvreauii group, Lactobacillus, Anaerorhabdus furcosa group, Victivallaceae, Desulfuromonadaceae, and Sediminispirochaeta. The predicted functional features represented by the Kyoto Encyclopedia of Genes and Genomes pathways were changed by LyJH307 toward a decrease of carbohydrate metabolism. Conclusions:LyJH307 caused a reduction of S. bovis and an increase of pH with shifts in minor microbiota and its metabolic pathways related to carbohydrate metabolism. This study provides the first insight into the availability of endolysin as a specific modulator for rumen and shows the possibility of endolysin degradation by rumen microbiota.