Hyperhomocysteinemia is a risk factor of cardiovascular disease (CVD). High-density lipoprotein (HDL) plays an important role in anti-atherosclerosis, with its anti-atherogenic function attributed to HDL-associated proteins such as apolipoprotein A-I (apoA-I) and paraoxonase 1 (PON1). Homocysteine (Hcy) thiolactone modifies lysine residues in proteins, thereby altering their function. Although dysfunction of apoA-I and PON1 has been reported, the precise modification sites and underlying mechanisms have remained unclear. In this study, we aimed to identify Hcy-thiolactone modification sites on apoA-I and PON1. In addition, we sought to clarify the effects of Hcy-thiolactone on PON1 activity and its distribution. Modification sites were analyzed using MALDI-TOF MS. The effects of Hcy-thiolactone on various specimens, including purified proteins, reconstituted HDL (rHDL), HDL collected by ultracentrifugation, and serum samples, were characterized using enzymatic assays measuring three major PON1 activities (arylesterase, paraoxonase, and lactonase) and Western blotting. Our results demonstrated that while some Hcy-thiolactone modification sites were detected on apoA-I, PON1 itself was not directly modified by Hcy-thiolactone. Thiolactonase activity was reduced by Hcy-thiolactone in large HDL particles. Furthermore, a general reduction of PON1 activity and changes in HDL remodeling and distribution were observed in serum samples treated with Hcy-thiolactone. These findings suggest that PON1 dysfunction induced by Hcy-thiolactone is influenced by alterations in HDL remodeling and the enzyme’s distribution on HDL particles. Analysis of PON1’s distribution dynamics under pathological conditions may provide crucial insights into the mechanism of HDL function decline in CVD.