The effects of nitroglycerine (NG) on the rumen methane emission, fermentation, and microbial community of Hu sheep were investigated. Eight sheep were fed NG (100 mg/head/day); another eight sheep served as controls. NG decreased methane emission of Hu sheep by ~ 19.3% (P < 0.05) without adversely affecting the production performance or rumen fermentation (P > 0.05). The alpha and beta diversity indexes of the bacterial and archaeal community showed no significant differences (P > 0.05). The dominant methanogenic species was the Methanobrevibacter gottschalkii clade, accounting for ~ 60%, followed by the Methanobrevibacter boviskoreani and Methanobrevibacter ruminantium clades. Prevotella 1 was the most dominant bacterial genus, accounting for ~ 42%, followed by the Rikenellaceae RC9 and Bacteroidales BS11 gut groups. In addition, pearson correlation analysis showed a few Methanomassiliicoccales species significantly correlated with several bacterial genera (P < 0.05).