Cyanocobalamin was found to be utilized as the precursor of DBCC by Propionibacterium shermanii as well as hydroxocobalamin, when added to the culture medium. Based upon the results, more useful method for preparing DBCC was established. It consists of cultivating Pr. shermanii in the presence of cyanocobalamin, harvesting the cells by centrifugation, extracting DBCC by homogenizing the cell paste with absolute alcohol, concentrating the extracts under reduced pressure, chromatographic separation on column of DEAE-cellulose or P-cellulose, treatment with phenol and crystallization by addition of acetone.