In sheep, temperament is known to affect animal welfare and the quality of animal products. While the composition of the gut microbiota is different between depressed patients and healthy human patients, in sheep, the influence of temperament on ruminal microbial species and abundance remains unknown. This study investigated the effects of temperament on parameters of rumen fermentation and microbial composition of rumen contents of Hu ram lambs. Using the pen score test, 6 lambs that scored 2 points or below (calm) and 6 lambs that scored 4 points or more (nervous) were selected from 100 ram lambs. The sheep were fed a standard diet for 60 days and rumen samples were collected at slaughter. The concentrations of propionic acid, isovaleric acid, valeric acid, and the ammonia nitrogen concentration were different between the calm and the nervous groups (p < 0.05). At the phylum level, there were significant differences in Bacteroidetes, Tenericutes, and Spirochetes (p < 0.05); and at the genus level, there were significant differences in the Christensenellaceae R-7 group, Treponema 2, Fibrobacter, and Ruminococcaceae UCG-003 (p < 0.05). The present study suggests that differences in the rumen microbiota between the calm group and the nervous group could have an impact on the metabolism of carbohydrates and polysaccharides and explain why Calm Hu sheep have a higher energy utilization efficiency than nervous Hu sheep. More studies are needed to further understand the effect of temperament on specific pathways of the rumen microbiota.