In order to provide more alternative epoxide hydrolases for industrial production, a novel cDNA geneRpeh-encoding epoxide hydrolase (RpEH) ofRhodotorula paludigenaJNU001 identified by 26S rDNA sequence analysis was amplified by RT-PCR. The open-reading frame (ORF) ofRpehwas 1236 bp encodingRpEH of 411 amino acids and was heterologously expressed inEscherichia coliBL21(DE3). The substrate spectrum of expressedRpEH showed that the transformantE. coli/Rpehhad excellent enantioselectivity to2a,3a, and5a-10a, among whichE. coli/Rpehhad the highest activity (2473 U/g wet cells) and wonderful enantioselectivity (E= 101) for8a, and its regioselectivity coefficients, alpha(R)and beta(S), toward (R)- and (S)-8awere 99.7 and 83.2%, respectively. Using only 10 mg wet cells/mL ofE. coli/Rpeh, the near-perfect kinetic resolution ofrac-8aat a high concentration (1000 mM) was achieved within 2.5 h, giving (R)-8awith more than 99% enantiomeric excess (ee(s)) and 46.7% yield and producing (S)-8bwith 93.2%ee(p)and 51.4% yield with high space-time yield (STY) for (R)-8aand (S)-8bwere 30.6 and 37.3 g/L/h.