In this study, we carried out a classification survey on sterile plants in the F₁ population of high-quality and high-yield Dian-type japonica hybrid rice ‘Dianheyou 34’, combining with the basic agronomic traits and biological information of the backbone restorer line ‘Nan34’ and its mutant Rf1 restorer gene lost mutant, the phenomenon and cause of function loss of restorer lines were preliminarily explored. The results showed that the loss of function with natural mutation at Rf1 in ‘Nan34’ was the main factor that led to sterile plants (two years accounted for 0.26% and 0.79% respectively) in ‘Dianheyou 34’F₁ hybrid population; the seed setting rate of ‘Nan34’ wild type was no significant difference compared with its mutant, and the wild type was slightly higher than the mutant in pollen fertility, plant height, ear length and grain length, and slightly greater than the wild type in grain width. By cloning the Rf1 locus of ‘Nan34’ wild type and its mutant, we found that the mutant in ORF region lost a 574 bp long sequence compared with the wild type, which made it unable to encode some PPR proteins. The loss of this sequence may lead to the loss of recovery function. The results lay a theoretical foundation for revealing genetic variation mechanism of restorer genes in three-line hybrid rice breeding and utilization.