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24 nt-siRNAs are the most abundant small interfering RNAs in rice grains aside from microRNAs. To investigate the roles that 24 nt-siRNAs played in the poor grain filling of rice inferior grains, dynamic variations of 24 nt-siRNAs in inferior grains were compared with those of superior grains by using small RNA deep sequencing technology. The results showed that 24 nt-siRNAs derived from multiple regions of rice genome, and the maintenance of the two strands of 24 nt-siRNA duplex was a non-random process. The amounts of 24 nt-siRNAs declined with the process of grain filling in both superior and inferior grains, but 24 nt-siRNAs in inferior grains was much higher than that of superior grains in each period we sampled. Bioinformatics prediction indicated that 24 nt-siRNAs targeted on more genes involved in most of the known KEGG rice pathways, such as the starch and sucrose biosynthesis pathway. Combined with digital gene expression profiling of target genes, 24 nt-siRNAs mapped on the antisense strands of exons were specifically investigated, but the abundance of 24 nt-siRNAs did not show negative correlations with their corresponding target genes. The results indicated that 24 nt-siRNAs were not involved in down-regulation of target genes. The potential biological meanings for this inconsistency were probably the results of methylation directed gene expression activation, or competition for small RNA stability methylation.