Methane is a short-lived greenhouse gas but has a far greater warming effect than carbon dioxide. At the same time, the livestock sector serves as a large contributor to global emissions of anthropogenic methane. Herein, this work aimed to use cultivated seaweed supplementation to reduce methane emissions and investigate the potential influencing mechanism. To evaluate the feasibility, two cultivated seaweeds, Laminaria japonica Aresch, and Porphyra tenera, along with the enzymatic hydrolysates derived from L. japonica, underwent in vitro trials, and they were both added into corn silage feed (CSF) with different concentrations (1%, 5%, and 10% of CSF) for methane reduction evaluation. The results indicated that >75% and 50% reductions in methane production were observed for the seaweeds and seaweed enzymatic hydrolysates in 9- and 30-day, respectively. Combined high-throughput sequencing and multivariate analysis revealed that supplementation with seaweed and seaweed enzymatic hydrolysates had a notable impact on the prokaryotic community structure. Mantel tests further revealed that significant correlations between the prokaryotic community and methane accumulation (P < 0.05), implying the prokaryotic community plays a role in reducing methane emissions within the rumen. Correspondingly, the networks within the prokaryotic community unveiled the crucial role of propionate/butyrate-producing bacteria in regulating methane emissions through microbial interactions. The predicted function of the prokaryotic community exhibited a significant reduction in the presence of the narB gene in seaweed-supplemented treatments. This reduction may facilitate an increased rate of electron flow toward the nitrate reduction pathway while decreasing the conversion of H2 to methane. These results indicated the supplementation of cultivated seaweeds and the enzymatic hydrolysates has the potential to reshape the community structure of rumen microbial communities, and this alteration appears to be a key factor contributing to their methane production-reduction capability.