C-repeat/dehydration-responsive element binding factors (CBF/DREBs) are a family of transcription factors that regulate freezing tolerance in Arabidopsis. As a step towards understanding the stress response of monocotyledonous plants, we isolated a barley gene HvCBF4 whose expression is induced by low-temperature stress. Transgenic over-expression of HvCBF4 in rice resulted in an increase in tolerance to drought, high-salinity and low-temperature stresses without stunting growth. Interestingly, under low-temperature conditions, the maximum photochemical efficiency of photosystem II in the dark-adapted state (Fv/Fm, where Fv is the variable fluorescence and Fm is the maximum fluorescence) in HvCBF4 plants was higher by 20% and 10% than that in non-transgenic and CBF3/DREB1A plants, respectively. Using the 60K Rice Whole Genome microarray, 15 rice genes were identified that were activated by HvCBF4. When compared with 12 target rice genes of CBF3/DREB1A, five genes were common to both HvCBF4 and CBF3/DREB1A, and 10 and seven genes were specific to HvCBF4 and CBF3/DREB1A, respectively. Interestingly, HvCBF4 did not activate Dip1 and Lip5, two important target genes of CBF3/DREB1A, in transgenic rice under normal growth conditions, but their expression was enhanced by HvCBF4 under low-temperature conditions. Our results suggest that CBF/DREBs of barley act differently from those of Arabidopsis in transgenic rice.