All organisms have a biological clock system which is strongly tied to how well an organism digests food and develops. This study aimed to understand the effects of circadian rhythm and feeding modes on rumen fermentation and microorganisms in Hu sheep. Forty-five healthy Hu sheep were randomly divided into three treatment groups of 15 sheep in each group, wherein they were fed the same concentrate and roughage. Under the condition that the nutrient-feeding amount was consistent throughout the day, the concentrate-to-forage ratio was dynamically adjusted during the day and night. Rumen fluid collected after the feeding experiment was used to determine the study parameters; the results showed a connection between rumen fermentation and the circadian clock. Volatile fatty acids (VFAs), pH, and NH3-N were significantly influenced by the fermentation duration (p 3-N concentration (p < 0.05). Under this feeding pattern, the activities of major digestive enzymes in the rumen, such as amylase and lipase, were significantly increased (p < 0.05), and the microbial diversity was also improved.