AbstractAimsThis study aims to investigate the effect of hydroxy-selenomethionine supplementation on the in vitro rumen fermentation characteristics and microorganisms of Holstein cows.Methods and ResultsFive fermentation substrates, including control (without selenium supplementation, CON), sodium selenite supplementation (0.3 mg kg−1 DM, SS03), and hydroxy-selenomethionine supplementation (0.3, 0.6 and 0.9 mg kg−1 DM, SM03, SM06 and SM09, respectively) were incubated with rumen fluid in vitro. The results showed that in vitro dry matter disappearance and gas production at 48 h was significantly higher in SM06 than SM03, SS03 and CON; propionate and total volatile fatty acid (VFA) production was higher in SM06 than CON. Moreover, higher species richness of rumen fluid was found in SM06 than others. Higher relative abundance of Prevotella and Prevotellaceae-UCG-003 and lower relative abundance of Ruminococcus-1 were detected in SM06 than CON. Besides, higher relative abundance of Ruminococcaceae_UCG-005 was found in CON than other treatments.ConclusionsIt is observed that 0.6 mg kg−1 DM hydroxy-selenomethionine supplementation could increase cumulative gas production, propionate, and total VFAs production by altering the relative abundance of Prevotella, Prevotellaceae-UCG-003, Ruminococcaceae_UCG-005 and Ruminococcus-1, so that it can be used as a rumen fermentation regulator in Holstein cows.Significance and Impact of the StudyThis study provides an optimal addition ratio of hydroxy-selenomethionine on rumen fermentation and bacterial composition via an in vitro test.