The D-psicose 3-epimerase (DPE) gene from Ruminococcus sp. was cloned and overexpressed in Escherichia coli. The recombinant protein was purified and characterized. It was optimally active at pH 7.5-8.0 and 60 °C. Activity was not dependent on the presence of metal ions; however, it became more thermostable with added Mn(2+). The K (m) of the enzyme for D-psicose (48 mM) was lower than that for D-tagatose (230 mM), suggesting that D-psicose is the optimum substrate. More importantly, the thermostability of the novel DPE from Ruminococcus is the strongest among all of the D-psicose and D-tagatose 3-epimerases and may be suitable for the industrial production of D-psicose from fructose.