Heterosis is a phenomenon that hybrids exhibit superior performance relative to the parental phenotypes. However, most studies focused on aboveground agronomic traits. Thus, systematic investigation on root heterosis, particularly at reproductive stage in rice is needed to be carried out. The recent advent of RNA sequencing technology (RNA-Seq) provides an opportunity to conduct in-depth transcript profiling for heterosis study.Using the Illumina HiSeq 2000 platform, the root transcriptome of a super-hybrid rice Xieyou9308 and its parents were analyzed at tillering and heading stages. Totally, 829 and 4186 differentially expressed genes between the hybrid and its parents (DGHP) were detected at tillering stage and heading stage, respectively. In this study, the DGHP were significantly enriched in pathways such as carbohydrate metabolism and plant hormones signal transduction at heading stage, and most of the key genes involved in the two pathways were up-regulated in the hybrid. Several significant DGHP that could be mapped to QTLs for yield and root traits were also involved in carbohydrate metabolism and plant hormones signal transduction pathways. The candidate transcripts may underlie the heterosis and significantly contribute to root development and yield production. Overall design: Root mRNA profiles of a super-hybrid rice Xieyou 9308 and its parents at tillering and heading stages were generated by deep sequencing, in duplicate, on Illumina Hiseq 2000 platform.