The root system is a critical component of rice plant， playing essential roles in plant fixation， water uptake， and nutrient acquisition. In this study， we observed that the root system of the oself3-1 mutant was significantly shorter compared to the wild type. To investigate the genetic basis of this trait， the mutant was crossed with the wild-type cultivar Sasaishiki to generate an F₂ population. Genetic analysis revealed that the mutant phenotype is controlled by a recessive single gene. Through map-based cloning， OsELF3-1 was located in a 50.9 kb interval on chromosome 6， which contains four open reading frames （ORFs）. Sequence analysis identified a 7-base pair deletion in the second exon of ORF4 （OsELF3-1）， resulting in frameshift mutation and premature termination. OsELF3-1 was hypothesized as the causal gene. Knockout mutants of OsELF3-1 exhibited significantly shorter roots the wild type Sasaishiki， thereby validating the role of OsELF3-1 in regulating root length. To further elucidate the regulatory network of OsELF3-1， we employed a yeast two-hybrid screening and identified OsARID3 as an interacting protein. OsARID3 contains several functional domains， including an ARID domain， an α-crystallin/Hsp domain， a HsP20 domain， and a potassium dependent sodium/calcium ion exchange domain. Investigation of OsARID3-RNAi mutants revealed showed that their mutants also displayed significantly shorter roots than wild type Sasaishiki， suggesting that OsARID3 is involved in the regulation of root length. Analysis of natural variation in OsARID3 using 3K rice genome sequencing data identified 15 haplotypes， with distinct differentiation between indica and japonica subspecies. Notably， japonica rice predominantly harbored haplotypes I， IV and VI. In summary， this study found that OsELF3-1 and OsARID3 interact to regulate rice root development. These findings provide a theoretical basis and valuable germplasm resources for further exploration of the regulatory networks underlying root growth and development.