[Background] D-mannose with many functional activities has been widely used in food, medicine, feed, etc. D-mannose isomerase can catalyze the reversible reaction between D-fructose and D-mannose, and has application potential in the enzymatic preparation of D-mannose. [Objective] The D-mannose isomerase gene (ssMIaseA) from Streptomyces sp. was cloned and expressed in Escherichia coli, and its enzymatic properties were studied and used to prepare D-mannose. [Methods] The D-mannose isomerase gene (ssMIaseA) from Streptomyces sp. was cloned and the recombinant expression plasmid pET-28a-ssMIaseA was constructed and expressed into E. coli BL21(DE3). After purification by Ni-NTA affinity chromatography, the enzyme properties were determined, and the preparation of D-mannose from SsMIaseA was analyzed by high performance liquid chromatography. [Results] SsMIaseA shared the highest homology of 60.2% with ManI from Thermobifda fusca. The specific activity of the enzyme was 525 U/mg, and the molecular weight was about 45 kD. Its optimal pH and temperature were 7.5 and 45 °C, respectively. It was stable in the range of pH 6.5?10.0 and below 45 °C. It had the highest catalytic activity for mannose, followed by D-fructose, D-talose and D-tagatose. SsMIaseA was used to convert 600 g/L D-fructose and the reaction reached equilibrium at 8 h, producing 185 g/L D-mannose with a conversion rate of 31%. [Conclusion] SsMIaseA as a new D-mannose isomerase has potential in the enzymatic preparation of D-mannose.