A cephalosporin acetylhydrolase gene, a recombinant DNA molecule prepared by introducing said gene into a vector used in an E. coli host-vector system, E. coli cells transformed with said recombinant DNA molecule, a protein having the amino acid sequence encoded by said gene, an enzyme which is a multimer of said protein, and a process for preparing said protein or enzyme are provided, said cephalosporin acetylhydrolase being an enzyme useful for converting cephalosporins such as cephalosporin C and 7-ACA into deacetylated ones such as deacetylcephalosporin C and deacetyl 7-ACA which are useful as an intermediate for preparing a variety of derivatized cephalosporin antibiotics.