Protein N-homocysteinylation involves a post-translational modification by homocysteine (Hcy)-thiolactone. In humans, about 70% of circulating Hcy is N-linked to blood proteins, mostly to hemoglobin and albumin. It was unclear what protein site(s) were prone to Hcy attachment and how N-linked Hcy affected protein function. Here we show that Lys⁵²⁵ is a predominant site of N-homocysteinylation in human serum albumin in vitro and in vivo. We also show that the reactivity of albumin lysine residues, including Lys⁵²⁵, is affected by the status of Cys³⁴. The disulfide forms of circulating albumin, albumin-Cys³⁴-S-S-Cys and albumin-Cys³⁴-S-S-Hcy, are N-homocysteinylated faster than albumin-Cys³⁴-SH. Although N-homocysteinylations of albumin-Cys³⁴-SH and albumin-Cys³⁴-S-S-Cys yield different primary products, subsequent thiol-disulfide exchange reactions result in the formation of a single product, N-(Hcy-S-S-Cys)-albumin-Cys³⁴-SH. We also show that N-homocysteinylation affects the susceptibility of albumin to oxidation and proteolysis. The data suggest that a disulfide at Cys³⁴ of albumin promotes conversion of N-(Hcy-SH)-albumin-Cys³⁴-SH to a proteolytically sensitive form N-(Hcy-S-S-Cys)-albumin-Cys³⁴-SH, which would facilitate clearance of the N-homocysteinylated form of mercaptoalbumin.