The objective of this experiment was to investigate the effects of 5,6-dimethylbenzimidazole (5,6-DMB) and cobalt (Co) on the ruminal vitamin B12 synthesis and fermentation parameters of sheep under high concentrate conditions. Twenty-four Kazakh rams (body weight = 39.23 ± 2.61 kg and 8 months old) fitted with permanent ruminal fistulas were randomly divided into four groups with six rams in each group. The control (CON) group was fed a basal ration with a concentrate-to-roughage ratio of 70:30, and the experimental groups (T60, T75 and T90) were fed a basal diet with 60 mg 5,6-DMB+ 0.25 mg Co, 75 mg 5,6-DMB+ 0.5 mg Co and 90 mg 5,6-DMB+ 0.75 mg Co supplied to each kilogram of basal diet, respectively. The experiment lasted for 26 days, with the first 14 days being an adaptation period to allow the sheep to adapt to the diet type and environment, and the second 12 days being a sample period. On 0, 7 and 12 d of the sample period, all sheep were weighed before the morning feed. Rumen fluid samples were collected from all sheep on the last 4 days of the sample period. The results showed that the ruminal vitamin B12 content was higher in trial groups than that of the CON group at 3 h after feeding (p < 0.05). Rumen pH was higher in trial groups than in the CON group at 1, 3 and 5 h after feeding (p < 0.05). The concentration of ruminal ammonia-N was significantly increased in trial groups when compared to the CON group (p < 0.05) at 1 and 3 h after feeding. At 1 and 3 h after feeding, the concentration of propionate in trial groups was higher than that in the CON group (p < 0.05). The rumen microbial protein content reached the highest value at 3 h after feeding, and all trial groups were higher than the CON group (p < 0.05). Thus, the supplementation of 5,6-DMB and Co increased vitamin B12, propionate, ammonia-N and microbial protein contents and pH in the rumen of sheep, and the best results were obtained by the amounts of 75 mg/kg 5,6-DMB and 0.5 mg/kg Co, respectively.