Root exudate metabolites are a key medium for the interaction between plants and soil microbiota. L-theanine is a unique non-protein amino acid critical for the flavor and potential health benefits of tea products; however, its biological function in tea plants is not well understood. As L-theanine is mainly synthesized in the roots of tea plants, we hypothesized that L-theanine could affect the function of the rhizosphere microbiota by modulating microbial assembly. In the present study, L-theanine was detected in the exudates of tea plant roots using liquid chromatography-mass spectrometry. Additionally, 16S rRNA gene sequencing revealed that L-theanine significantly altered the structure of the rhizosphere microbiota and selectively shaped rhizosphere microbial assembly. Moreover, metagenomic data showed that L-theanine affected the abundance of genes encoding element cycling in soil. Interestingly, the denitrification and complete nitrification pathways were significantly inhibited by L-theanine by decreasing the narH, napA, and napB genes abundance. These findings provide new insights into the biological function of L-theanine, as well as the implications of interactions between tea plant root exudates and the rhizosphere microbiome.