Xylose-utilizing and thermophilic Geobacillus sp. Y565-5 was isolated from surface soil of an oilfield in Yumen Town, Gansu Province, China. A xylose isomerase (XylA) gene was cloned from the strain by PCR. The open reading frame of xylA (1 182 bp) encoded a protein of 394 amino acids, which showed high sequence homology (99% identity) with that of Geobacillus sp. Y412MC52. The intact coding region was subcloned into pET28a(+) vector and expressed in Escherichia coli BL21(DE3). The molecular weight of the recombinant protein was 45 kD based on SDS-PAGE and its xylose isomerase activity was detected through cysteine welts thiazole method after the induction of isopropyl β-D-1-thiogalactopyranoside? (IPTG). The optimum temperature and pH for the partially purified recombinant XylA activity were 90 °C and pH 8.0, respectively.