Plants have a large family of HSFs with different roles in the heat shock response that mediate the expression of HSP regulated genes. The HSF encoding genes are easily identified by their highly conserved modular structure and motifs. In the present study, a putative GmHsfA1 was identified and characterized from the soybean expressed sequence tag (EST) database by sequence comparison with the functionally well-characterized LpHsfA1 and rapid amplification of cDNA ends (RACE). Multiple alignment showed that the amino acid sequence of GmHSFA1, matching best with LpHSFA1 (52.2% similarity), was obviously different from that of each of several HSFA1s from other plant species. The GmHsfA1 has a constitutive expression profile in the different tissues examined. The overexpression of GmHsfA1 in transgenic soybean plants led to the activation of GmHsp70 under normal temperature and the overexpression of GmHsp70 under high temperature. Furthermore, transgenic soybean plants with GmHsfA1 overexpression showed obvious enhancement of thermotolerance under heat stress in comparison with non-transgenic plants. The experimental results suggested that GmHSFA1 is a novel and functional heat-shock transcription factor.