The effects of dietary supplementation of HMBi, alone or in combination with B vitamins were investigated on fermentation characteristics and in vitro ruminal and post-ruminal digestion using three modified techniques. A total-mixed ration was formulated using the assumptive animal information by CPM-Dairy software and used as a substrate. Eight dietary treatments arranged in a 2×2×2 factorial design with factors of HMBi (0 and 0.13% DM), folic acid (0 and 0.012% DM), and vitamin B12 (0 and 0.0021% DM). In the first experiment, fermentation characteristics and parameters were evaluated for dietary supplements’ effects using in vitro gas measuring technique. In the second experiment, an in vitro modified batch culture technique was performed to study on in vitro digestion and fermentation end products. Finally, ruminal and post-ruminal digestibility of crude protein was investigated by a modified three-step technique. In comparison with control, supplementation of HMBi declined cumulative gas production (P < 0.001) and fermentation parameters (P < 0.01) all over the incubation times. Interaction effects between B9 and B12 were identified (P ≤ 0.01) that were associated with the decreased cumulative gas production (P ≤ 0.01), A fraction (P = 0.01) and C fraction (P = 0.02). Digestion of CP declined at first incubation time by alone supplementation of HMBi, folic acid and B12 but it was improved after that. Supplementation of folic acid alone or in combination with HMBi increased (P≤0.05) NDF digestion for 4 and 24 h of incubation. Ammonia-N was decreased significantly with supplementation compared to intact diet. Total VFA was also enhanced (13 to 46%) by dietary supplementation of B12 compared to unsupplemented diet. Highest VFA was associated with HMBi from 24 h and thereafter. Addition of HMBi individually or with B9 and B12 increased post-ruminal CP while B vitamins had not the same effect of supplementing individually. Results suggest that supplementation of diet with HMBi and B vitamins, modify rumen fermentation by decreasing gas production, enhancing production of VFA and increasing utilization of ammonia-N in the rumen

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