Abstract: l-Arabinose isomerase (EC 220.127.116.11, l-AI) mainly catalyzes the reversible aldose–ketose isomerization between l-arabinose and l-ribulose. l-AIs can also catalyze other reactions, such as the conversion of d-galactose to d-tagatose. In this study, the araA gene encoding l-AI was PCR-cloned from Thermoanaerobacterium saccharolyticum NTOU1 and then expressed in Escherichia coli. The recombinant l-AI was purified from the cell-free extract using nickel nitrilotriacetic acid metal-affinity chromatography. The purified enzyme showed an optimal activity at 70 °C and pH 7–7.5. The enzyme was stable at pHs ranging from 6.5 to 9.5 and the activity was fully retained after 2 h incubation at 55–65 °C. The low concentrations of divalent metal ions, either 0.1 mM Mn2+ or 0.05 mM Co2+, could improve both catalytic activity and thermostability at higher temperatures. The recombinant T. saccharolyticum NTOU1 l-AI has the lowest demand for metal ions among all characterized thermophilic l-AIs. This thermophilic l-AI shows a potential to be used in industry to produce d-tagatose from d-galactose.