Objectives Establish a complete and efficient method for the preparation ofcis-5-hydroxy-L-pipecolic acids (cis-5HPA), including biotransformation and isomers separation and purification. Results For non-heme Fe(II)/alpha-KG-dependent dioxygenases, alpha-ketoglutarate (alpha-KG) has great influence on the stability of Fe(II) ions, which is also the basic of the hydroxylation reaction to the substrate. L-pipecolic acids (L-Pip) was converted tocis-5HPA by whole-cell catalysis in water, which can reduce the loss of Fe(II) ions. 120 mM L-Pip can be transformed to 93% via cell and Fe(II) ions continuous supplementation under the reaction system optimization (the molar ratio of ascorbic acid/FeSO4 center dot 7H(2)O and alpha-KG/L-Pip were 8:1 and 1:1, respectively). After the catalytic reaction, the amino protection strategy was adopted to improve the resolution of isomer products on silica gel chromatography, and the amino protectedcis-5HPA was obtained with a yield of 86.7%. Conclusions We established a method which is promising to be used forcis-5HPA largescale preparation. It also provides a suitable reference for this type of enzyme-catalyzed reaction and the hydroxy pipecolic acid isomers separation.