The effects of incorporating yeast culture (YC) into pelleted feeds on sheep production and the potential impact on rumen microbial populations, microbial metabolism, and fermentation have not been extensively studied. This study aimed to evaluate the effect of YC on growth performance, rumen tissue development, rumen fermentation, and rumen microflora in sheep and to explore the potential microbial mechanisms involved. Fifty healthy 3-month-old male lambs of small-tailed Han sheep, with an average weight of 28.44 ± 0.63 kg, were randomly divided into five groups: control (0% YC), 3% YC, 6% YC, 9% YC, and 12% YC. The pre-feeding period lasted for 15 days, followed by an official feeding period of 60 days. On the last day of the formal feeding period, six lambs that exhibited the best growth performance were randomly selected from the control group and the 9% YC group. These sheep were slaughtered, then the rumen epithelial tissue and rumen contents were collected for the measurement of rumen fermentation, microbial populations, and metabolites. Compared to the control group, the YC-treated groups showed higher daily and final body weight gains, as well as increased levels of propionic acid, butyric acid, and total volatile fatty acids (