Recombinant human deoxyribonuclease I (rhDNase) is the mucolytic agent most widely used for the treatment of respiratory disease in cystic fibrosis. However, rhDNase is rapidly cleared from the lungs which implies a high dosing frequency and limited patient adherence. The aim of this study was to produce a long-acting PEGylated derivative of rhDNase presenting a preserved enzymatic activity. Site-specific PEGylation on the N-terminal (N-ter) leucine residue of rhDNase was achieved by reductive alkylation at acidic pH using linear 20 kDa, linear 30 kDa or two-arm 40 kDa polyethylene glycol (PEG) propionaldehydes. Yields of mono-PEGylated products ranged between 45% and 61%. Conjugation to PEG fully preserved the secondary structure and the in vitro enzymatic activity of the native protein. These properties offer interesting perspectives for in vivo inhalation studies of the PEGylated enzyme.