L-isoleucine is produced by a fermentation process comprising aerobically culturing in an aqueous culture medium an L-isoleucine producing microorganism obtained by isolating a transformed strain resistant to α-amino-β-hydroxy valeric acid prepared by incorporating into a recipient strain of the genus Brevibacterium or Corynebacterium, which is sensitive to α-amino-β-hydroxy valeric acid, a plasmid DNA obtained from a microorganism of the genus Brevibacterium or Corynebacterium into which has been inserted a fragment of chromosomal DNA derived from a DNA-donor strain of the genus Brevibacterium or Corynebacterium which is resistant to α-amino-β-hydroxy valeric acid; and recovering L-isoleucine which accumulates in the resulting culture liquid.