Several strategies for site-specific PEGylation have been successfully exploited to conjugate poly(ethylene glycol) (PEG) to pharmaceutical proteins. The advantages sought are those of improving efficacy and increasing the half-life of conjugated proteins while achieving a higher degree of homogeneity. Recombinant human growth hormone (hGH) was thus PEGylated exploiting two site-specific strategies: N-terminal PEGylation using the PEG20 kDa-aldehyde polymer and microbial transglutaminase (mTGase) mediated enzymatic PEGylation using PEG20 kDa-NH2. N-Terminal PEGylation of hGH was carried out by covalent attachment of PEG to the α-amine residue of Phe1 that yielded the monoconjugate PEG-Nter-hGH with a mass of 44152.2 Da, as measured by MALDI-TOF mass spectrometry. The mTGase mediated PEGylation, performed in a water/ethanol solution mixture, allowed a PEG coupling reaction only at the level of hGH Gln141, yielding the single monoconjugate PEG-Gln141-hGH with a mass of 44064.9 Da. Circular dichroism studies showed that both conjugation strategies preserved the native-like secondary structures of hGH. It is vital to maintain the structural integrity of hGH if PEGylated hGH is to be used in therapeutic applications. As expected, the pharmacokinetic profile in rats of PEG-Nter-hGH and PEG-Gln141-hGH revealed a significant increase in systemic exposure with respect to unmodified hGH. The conjugates showed a half-life increase of 4.5-fold with respect to hGH. These results demonstrate that both chemical and enzymatic site-selective PEGylation of hGH generates conjugates with a prolonged half-life.