Rice (Oryza sativa) seedlings are particularly sensitive to chilling in early spring in temperate and subtropical zones and in high-elevation areas. Improvement of chilling tolerance in rice may significantly increase rice production. MYBS3 is a single DNA-binding repeat MYB transcription factor previously shown to mediate sugar signaling in rice. In this study, we observed that MYBS3 also plays a critical role in cold adaptation in rice. Gain- and loss-of-function analyses indicated that MYBS3 was sufficient and necessary for enhancing cold tolerance in rice. Transgenic rice constitutively overexpressing MYBS3 tolerated 4°C for at least 1 week and exhibited no yield penalty in normal field conditions. Transcription profiling of transgenic rice overexpressing or underexpressing MYBS3 led to the identification of many genes in the MYBS3-mediated cold signaling pathway. Several genes activated by MYBS3 as well as inducible by cold have previously been implicated in various abiotic stress responses and/or tolerance in rice and other plant species. Surprisingly, MYBS3 repressed the well-known DREB1/CBF-dependent cold signaling pathway in rice, and the repression appears to act at the transcriptional level. DREB1 responded quickly and transiently while MYBS3 responded slowly to cold stress, which suggests that distinct pathways act sequentially and complementarily for adapting short- and long-term cold stress in rice. Our studies thus reveal a hitherto undiscovered novel pathway that controls cold adaptation in rice.