The restoring ability of restorer lines plays an important role in the fertility stability of three line F1 hybrids. In this study, two soybean cytoplasmic male sterile F1 hybrids, which were produced with the same male sterile line pollinated by restorer lines showing strong and weak different restoring ability, respectively, were used. The transcriptome sequencing of mixed flower buds of different sizes was carried out at the flowering stage. Through comparative analysis, according to "p-value < 0.05 and | log2foldchange | > 1" as the threshold in F1 hybrid (weak restorer) if compared to that in F1 hybrid (strong restorer), 2060 differentially expressed genes (DEGs) were identified including 1446 and 614 DEGs were down-regulated and up-regulated, respectively. The transcripts of several genes using quantitative real-time PCR (qRT-PCR) analysis were coincident with the results of RNA sequencing (RNA-Seq). The significance enrichment analysis of gene ontology (GO) showed that the main differential biological functions were cell periphery, pectinesterase inhibitor activity, pectinesterase activity, cell wall and external packaging structure. The enrichment analysis of kyoto encyclopedia of genes and genomes (KEGG) pathway showed that the main differential metabolic pathways were pentose and glucuronate interconversions, plant pathogen interaction and glycolysis / gluconeogenesis. According to the results of differential transcriptional genes analysis and literature reports, it is speculated that the fertility stability of soybean cytoplasmic male sterile F1 hybrid was related to the genes involved in pollen cell wall development, carbohydrate metabolism and plant pathogen interaction. When the functional balance was challenged due to the changes of environmental conditions, the sterility and fertility would be switched. Collectively, this study provided valuable information for understanding the molecular mechanism of cytoplasmic male sterility and fertility restoration in soybean from the aspect of fertility stability.