Key message A candidate gene cytochrome b5 for the major QTL qSRMP9 for rice seed reserve mobilization was validated during seed germination using a genome-wide association study approach. Seed reserve mobilization plays important roles in the early seedling growth in rice. However, the genetic basis underlying this process is poorly understood. In this study, the genetic architecture of variation in seed reserve mobilization during seed germination was studied using a genome-wide association study approach in rice. Three quantitative trait loci (QTL) including qSRMP6, qSRMP9, and qSRMP12 for seed reserve mobilization percentage were identified. In which, the candidate gene cytochrome b5 (OsCyb5) for the major QTL qSRMP9 was validated. Disruption of this gene in Oscyb5 mutants reduced the seed reserve mobilization and seedling growth compared with wild-type (WT) in rice. There were no significant differences of grain size, starch, protein and total soluble sugar content in the mature grains between Oscyb5 mutants and WT. However, the alpha-amylase activity in the germinating seeds of Oscyb5 mutants was significantly decreased compared to that of WT, and then, the starch and sugar mobilization and the glucose accumulation during seed germination were significantly decreased in Oscyb5 mutants. Two elite haplotypes of OsCyb5 associated with the higher seed reserve mobilization percentage and its elite single nucleotide polymorphism variations were mainly existed in the INDICA and AUS accessions. The natural variation of OsCyb5 contributing to seed reserve mobilization might be useful for the future rice breeding.