Thyrogen (thyrotropin alfa for injection), recombinant human TSH (rhTSH), has been successfully used to enhance diagnostic radioiodine scanning and thyroglobulin testing in the follow-up of patients with thyroid cancer and as an adjunctive treatment for radioiodine thyroid remnant ablation. However, the short half-life of rhTSH in the circulation requires a multidose regimen. We developed novel sialic acid-mediated and galactose-mediated conjugation chemistries for targeting polyethylene glycol (PEG) to the three N-linked glycosylation sites on the protein, to prolong plasma half-life by eliminating kidney filtration and potential carbohydrate-mediated clearance. Conjugates of different PEG sizes and copy numbers were screened for reaction yield, TSH receptor binding, and murine phamacokinetics/pharmacodynamics studies. The best performing of these products, a 40-kDa mono-PEGylated sialic acid-mediated conjugate, exhibited a 3.5-fold longer duration of action than rhTSH in rats, as a 5-fold lower affinity was more than compensated by a 23-fold extension of circulation half-life. Biochemical characterization confirmed conjugation through the sialic acids. Correlation of PEG distribution on the three N-linked glycosylation sites and the PEG effect on receptor binding supported the previously reported structure-function relationship of rhTSH glycosylation. This long-acting rhTSH has the potential to significantly improve patient convenience and provider flexibility while reducing potential side effects associated with a sudden elevation of serum TSH.