Osteocalcin (OCN) is a hormone secreted specifically by osteoblasts, which favors insulin secretion and insulin sensitivity, and prevent sarcopenia and age-related memory loss. OCN functions are regulated by at least two post-translational modifications (PTMs): OCN carboxylation by the gamma-carboxylase inhibits its function, while in contrast, pro-OCN processing by the proprotein convertase furin is required for its maturation. Interestingly, we noticed that throughout lifespan, mice have 5- to 10-fold higher circulating level of OCN than humans. Based on this observation and other preliminary data, we hypothesize that an additional species-specific PTM is involved in the regulation of OCN circulating levels. To identify this PTM mouse OCN (mOCN) was immunoprecipitated from bone extracts and characterized by reverse phase HPLC followed by mass spectrometry and tandem mass spectrometry. These “top-down” proteomics analyses revealed that mOCN is subjected to O-glycosylation. Using cell-based and biochemical assays we confirmed that mOCN is O-glycosylated in vitro and in vivo, while site-directed mutagenesis identified a single N-terminal serine (Ser) residue as the O-glycosylation site in mOCN. We next examined whether O-glycosylation regulates other mOCN PTMs and investigated the requirement of O-glycosylation for mOCN function in vivo. Pharmacological inhibition of mOCN O-glycosylation, processing or gamma-carboxylation in osteoblast cultures revealed that O-glycosylation occurs independently of the other mOCN PTMs. However, we found that O-glycosylation increase mOCN half-life in plasma ex vivo as well as in vivo in mice. Surprisingly, the O-glycosylated Ser residue in mOCN corresponds to a tyrosine (Tyr) in human OCN (hOCN), and cell-based assays indicate that hOCN is indeed not O-glycosylated. Conversely, mutation of this Tyr to a Ser residue is sufficient to induce O-glycosylation of hOCN. Finally, O-glycosylated hOCN has increased half-life in plasma compare to native hOCN. Together these results uncover a novel species-specific PTM of OCN that may explain why mice have higher circulating level of this hormone than humans. In addition, we establish that a single amino acid change can confer to hOCN a longer half-life through its O-glycosylation, bearing important implications for the use of OCN in therapeutic applications for human diseases such as diabetes, age-related memory loss and sarcopenia.