Silicon (Si) has the potential to improve plant growth and stress tolerance. The study aimed to explore Si-involving plant responses and molecular characterization of different Si-responsive genes in alfalfa. In this study, the exogenous supplementation of Si enhanced plant growth, and biomass yield. Si-acquisition in alfalfa root and shoot was higher in Si-supplemented compared to silicon deficient (-Si) plants, implying Si-acquisition has beneficial on alfalfa plants. As a consequence, the quantum efficiency of photosystem II (Fv/Fm) was significantly increased in silicon-sufficient (+Si) plants. The quantitative gene expression analysis exhibited a significant upregulation of the Lsi1, Lsi2, Lsi3, NIP5;1, and NIP6;1 genes in alfalfa roots, while BOR1, BOR4, NIP2, and NIP3 showed no significant variation in their expression. The MEME results further noticed the association of four motifs related to the major intrinsic protein (MIP). The interaction analysis revealed that NIP5;1 and Lsi1 showed a shared gene network with NIP2, BOR1, and BOR4, and Lsi2, Lsi3 and NIP3-1, respectively. These results suggest that members of the major intrinsic proteins (MIPs) family especially Lsi1, Lsi2, Lsi3, NIP5;1, and NIP6;1 genes helped to pass water and other neutral solutes through the cell membrane and those played significant roles in Si uptake and transport in plants. Together, these insights might be useful for alfalfa breeding and genome editing approaches for alfalfa improvement.