Abstract: Human insulin or B-30 esters thereof are prepared by reacting an insulin derivative of the formula ##STR1## wherein R is hydroxyl or an amino acid radical being different from threonine, and -A- and -B- represent the A- and B-chains with the same amino acid sequence as in human insulin, with a carboxyl protected and optionally hydroxyl protected L-threonine derivative in a concentration of from 2 to about 6 moles per liter in the presence of trypsin or a trypsin-like enzyme in a reaction medium containing water and optionally also an organic cosolvent at a pH value of from 5 to 9 and at a temperature below 50.degree. C., followed, if desired, by removal of any protecting groups present.Human insulin is prepared in an easy and simple way in high yield and in high purity.