Abstract The objective of this study was to evaluate the effects of different concentrations of hydrogen-rich water (HRW) on in vitro rumen fermentation characteristics and the dynamics of bacterial communities. The experiment included four treatment groups: a control (CON) and hydrogen-rich water (HRW) at 200, 400, and 800 ppb. Each group was analyzed at 12-hour (h) and 48-hour (h) time points with five replicates, totaling 40 samples. The experimental results highlighted the HRW800ppb group as the top production in terms of gas production and CH4 content. In contrast, the HRW200ppb group exhibited significantly lower methane levels at both 12 h and 48 h (P 0.05). Although HRW did not significantly benefit the synthesis of total volatile fatty acids (TVFA) and individual VFA, the HRW800ppb group significantly increased the ratio of acetate to propionate (P