The root system is an important part of rice plant and plays an important role in plant fixation, water and nutrient acquisition. In this study, it was found that the root system of oself3-1 mutant became significantly shorter, the mutant was crossed with wild type to construct F2 isolated population, and genetic analysis showed that the mutant trait was regulated by recessive single gene. By mapping cloning, OsELF3-1 was located in a 50.9 kb interval of chromosome 6, in which there were four open reading frames (ORFs), sequence comparison revealed that the mutant had 7 bases missing on the second exon of ORF4 (OsELF3-1), causes the gene to frameshift and terminate prematurely, and OsELF3-1 was presumed to be the target gene. The root system of CRISPR/Cas9 mutant with OsELF3-1 was significantly shorter than that of wild type, which verified that OsELF3-1 was involved in regulating root length. In order to further clarify the regulatory network of OsELF3-1, the interacting protein OsARID3 of OsELF3-1 was screened by yeast two-hybridization, OsARID3 has an ARID functional domain, an α-crystallin/Hsp_domain, a SHSP domain, and a potassium dependent sodium/calcium ion exchange domain. Investigation of OsARID3-RNAi mutants showed that their roots were significantly shorter than those of the wild type, OsARID3 may be involved in the regulation of root length. Natural variation of OsARID3 was analyzed by 3 K sequencing data and 15 haplotypes were found, and there was obvious differentiation between indica and japonica, japonica rice mainly belonged to haplotypes I, IV and VI. In summary, this study found that OsELF3-1 and OsARID3 interact to regulate rice roots, providing theoretical basis and germplasm resources for further analysis of rice root growth and development regulation network.