Plants have a close relationship with their root microbiota, which comprises a complex microbial network. Histone methylation is an important epigenetic modification influencing multiple plant traits; however, little is known about the role of plant histone methylation in the assembly and network structure of the root microbiota. In this study, we established that the rice (Oryza sativa) histone methylation regulates the structure and composition of the root microbiota, especially the hub species in the microbial network. DJ-jmj703 (defective in histone H3K4 demethylation) and ZH11-sdg714 (defective in H3K9 methylation) showed significant different root microbiota compared with the corresponding wild types at the phylum and family levels, with a consistent increase in the abundance of Betaproteobacteria and a decrease in the Firmicutes. In the root microbial network, 35 of 44 hub species in the top 10 modules were regulated by at least one histone methylation-related gene. These observations establish that the rice histone methylation plays a pivotal role in regulating the assembly of the root microbiota, providing insights into the links between plant epigenetic regulation and root microbiota.