The dehydration-responsive element binding proteins (DREB1)/C-repeat (CRT) binding factors (CBF) function as transcription factors and bind to the DRE/CRT cis-acting element (core motif: G/ACCGAC) commonly present in cold-regulated (COR) genes and subsequently upregulate the expression of such genes in Arabidopsis. We identified a DREB1A/CBF3-like gene, designated LpCBF3, from perennial ryegrass (Lolium perenne L.) by using RT-PCR and RACE (rapid amplification of cDNA end). The LpCBF3 gene contains all the conserved domains known to exist in other CBF genes. A comprehensive phylogenetic analysis using known and computationally identified CBF homologs in this study revealed that all monocot CBF genes are separately clustered from eudicot CBF genes and the LpCBF3 is the ortholog of rice OsDREB1A/CBF3 gene. Similar to other DREB1A/CBF3 homologs, expression of the LpCBF3 is induced by cold stress, but not by abscisic acid (ABA), drought, or salinity. Overexpression of the LpCBF3 cDNA in Arabidopsis induced expression of the Arabidopsis DREB1A/CBF3 target COR genes, COR15a and RD29A, without cold acclimation. Ion leakage in leaves of the overexpression transgenic plants was significantly reduced, an indication of enhanced freezing tolerance. Our data demonstrated that LpCBF3 not only resembles DREB/CBF genes of Arabidopsis, but is also capable of functioning as a transcriptional regulator in Arabidopsis, a species distant to the grass family.