MYB transcription factors have been demonstrated to play an important role in plant growth, development and abiotic stresses. This study isolated a rice MYB gene, OsMYB1R1 (Os04g0583900), and functionally characterized its role in tolerance to drought stress by generating transgenic rice plants with overexpressing (OE) and RNA interference (RNAi) OsMYB1R1 . Expression of OsMYB1R1 was down-regulated by drought stress. The tissue-specific expression analysis indicated that OsMYB1R1 was expressed at high level in panicle, but relatively low in the other parts of rice. No difference in germination rate among OsMYB1R1- OE, RNAi and wild-type (WT) seeds under mannitol treatments. No differences in phenotypes, physiological indicators and agronomic traits among WT, OE and RNAi plants were observed under normal grown conditions. Under drought stress, the RNAi plants were more tolerant to drought stress and higher survival rate after re-watering than WT plants, whereas, the overexpressing plants have found just the opposite. The OsMYB1R1 -OE plants exhibited increased relative electrical conductivity (REC), increased malondialdehyde (MDA) content, and decreased proline content compared with the wild type, whereas lower REC and MDA content and higher proline content were found in the RNAi plants. These results suggest that OsMYB1R1 functions as a negative regulator in response to drought stresses, and may be used as a candidate gene for molecular breeding of drought-tolerant crop varieties.