Abstract: A process for preparing human insulin, which comprises the steps of: preparing a DNA encoding a human proinsulin derivative having the following formula (I); inserting the DNA into a vector to construct an expression vector for the expression of the human proinsulin derivative; transforming a host cell with the expression vector; culturing the resulting transformant under a condition that allows the expression of the human proinsulin derivative; purifying human proinsulin derivative from the culture; and preparing human insulin from the human proinsulin derivative by an enzymatic hydrolysis:B chain-Z-A chain (I)Wherein,said A and B chains are respectively human insulin chains;Z is a peptide of formula U-U-X-P-J-(X')n-U-U;U is an arginine or lysin residue;X and X' are independently any amino acid residue;P is a proline residue;J is a glycine, arginine or lysine residue; andn is 0 or an integer of 1 to 5.