Background: The potential impact of genetically modified (GM) plants on human health has attracted much attention worldwide, and the issue remains controversial. This is in sharp contrast to the broad acceptance of plants produced by breeding through Marker Assisted Backcrossing (MAB). Results: Focusing on transcriptome variation and perturbation to signaling pathways, we assessed the molecular and biological aspects of substantial equivalence, a general principle for food safety endorsed by the Food and Agricultural Organization and the World Health Organization, between a transgenic crop and a plant from MAB breeding. We compared a transgenic rice line (DXT) and a MAB rice line (DXB), both of which contain the gene Xa21 providing resistance to bacterial leaf blight. By using Next-Generation sequencing data of DXT, DXB and their parental line (D62B), we compared the transcriptome variation of DXT and DXB. Remarkably, DXT had 43% fewer differentially expressed genes (DEGs) than DXB. The genes exclusively expressed in DXT and in DXB have pathogen and stress defense functions. Functional categories of DEGs in DXT were comparable to that in DXB, and seven of the eleven pathways significantly affected by transgenesis were also perturbed by MAB breeding. Conclusions: These results indicated that the transgenic rice and rice from MAB breeding are substantial equivalent at the transcriptome level, and paved a way for further study of transgenic rice, e.g., understanding the chemical and nutritional properties of the DEGs identified in the current study.