Dioscorea opposite waste (DOW) has been shown to improve the gastrointestinal microbiome, antioxidation capacity, and immune activity, indicating it is a potential feed resource to improve the physiological health and rumen function of weaned lambs. In the present study, the responses of rumen microbiome to DOW supplementation in diet were profiled using metagenome sequencing. In addition, the potential of DOW to regulate plasma parameters in weaned lambs and its possible mechanisms were investigated. Sixty healthy male small tail Han lambs (22.68 ± 2.56 kg) were selected and equally assigned to four dietary treatments: (1) DOW-free diet (CON), (2) addition of 10% DOW diet (DOW1), (3) addition of 15% DOW diet (DOW2), and (4) addition of 20% DOW diet (DOW3). Experimental lambs were fed a corresponding diet for 62 days. Rumen microbiome and plasma parameters were determined at the end of the experiment. The results showed that dietary supplementation with DOW linearly increased the concentration of aspartate aminotransferase, alkaline phosphatase, Immunoglobulin A, Immunoglobulin M, Immunoglobulin G, Glutathione peroxidase, Superoxide dismutase, and total antioxidant capacity in the plasma of weaned lambs, but an opposite trend was observed in Interleukin-1β, Interleukin-6, tumor necrosis factor-α, and Malondialdehyde between the DOW-supplemented group and the CON group. Sequencing of rumen metagenome revealed that dietary supplementation with 20% DOW significantly affected the microbial composition and function and increased the richness and diversity of rumen microbiota and relative abundance of phylum Verrucomicrobia, Planctomycetes, Fibrobacteres, Chloroflexi, Actinobacteria, and Acidobacteria and species Ruminococcaceae_bacterium, Clostridiales_bacterium_NK3B98, Clostridiales_bacterium, and Clostridia_bacterium. It was concluded that supplementing the weaned lamb’s ration with DOW increased the immune response and antioxidant capacity in a dose-dependent manner. Meanwhile, dietary supplementation with 20% DOW modulated the composition of rumen microbiome function by increasing Ruminococcaceae_bacterium and Clostridiales_bacterium with improving the polysaccharide hydrolase activity in the rumen.