Rice (Oryza sativa L.) is one of the most important species for food production worldwide. Low temperature is a major abiotic factor that affects rice germination and reproduction. Here, the underlying regulatory mechanism in seedlings of a TGMS variety (33S) and a cold-sensitive variety (Nipponbare) was investigated by comparative transcriptome. There were 795 differentially expressed genes (DEGs) identified only in cold-treated 33S, suggesting that 33S had a unique cold-resistance system. Functional and enrichment analysis of these DEGs revealed that, in 33S, several metabolic pathways, such as photosynthesis, amino acid metabolism, secondary metabolite biosynthesis, were significantly repressed. Moreover, pathways related to growth and development, including starch and sucrose metabolism, and DNA biosynthesis and damage response/repair, were significantly enhanced. The expression of genes related to nutrient reserve activity were significantly up-regulated in 33S. Finally, three NAC and several ERF transcription factors were predicted to be important in this transcriptional reprogramming. This present work provides valuable information for future investigations of low-temperature response mechanisms and genetic improvement of cold-tolerant rice seedlings.