OBJECTIVE To isolate, purify and identify the structures of related impurities from biotransfomated calcifediol. METHODS After vitamin D3爁ermentation broth was crudely extracted, high-pressure preparative chromatography was adopted to isolate calcifediol repeatedly, acquiring high purity related impurities from calcifediol. When the impurities reached a certain purity, through DAD Scanning, Mass Spectrometry and Nuclear Magnetic Resonance, the structure of chemical compound was identified. RESULTS After purification, 97.5% purity of impurity item was obtained. After structure identification, the impurity is (5Z,7E)-9,10-ring opening 5,7,10(19)-triene-3b,24-diol which means that 24-hydroxy vitamin D3 is the isomer of calcifediol. CONCLUSION The results of this study provide the basis for the establishment of quality standard of biotransfomated calcifediol as active pharmaceutical ingredients. Meanwhile, they also provide impurity reference for subsequent experiental work.