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IntroductionExtreme environments at high altitudes pose a significant physiological challenge to animals. We evaluated the gut microbiome and fecal metabolism in Sanhe heifers from different altitudes.MethodsTwenty Sanhe heifers (body weight: 334.82 ± 13.22 kg, 15-month-old) selected from two regions of China: the Xiertala Cattle Breeding Farm in Hulunbeier, Inner Mongolia [119°57′ E, 47°17′ N; approximately 700 m altitude, low altitude (LA)] and Zhizhao Dairy Cow Farm in Lhasa, Tibet [91°06′ E, 29°36′ N; approximately 3,650 m altitude, high altitude (HA)], were used in this study. Fecal samples were collected and differences in the gut microbiota and metabolomics of Sanhe heifers were determined using 16S rRNA gene sequencing and metabolome analysis.Results and discussionThe results showed that altitude did not significantly affect the concentrations of fecal volatile fatty acids, including acetate, propionate, butyrate, and total volatile fatty acids (p > 0.05). However, 16S rRNA gene sequencing showed that altitude significantly affected gut microbial composition. Principal coordinate analysis based on Bray–Curtis dissimilarity analysis revealed a significant difference between the two groups (p = 0.001). At the family level, the relative abundances of Peptostreptococcaceae, Christensenellaceae, Erysipelotrichaceae, and Family_XIII were significantly lower (p |