Phthalate esters (PAEs) are globally pervasive contaminants that are considered to be endocrine disruptor chemicals and toxic environmental priority pollutants. In this paper, the interactions between PAEs and human serum albumin (HSA) were examined by molecular modelling, steady state and time-resolved fluorescence, ultraviolet-visible spectroscopy (UV-vis) and circular dichroism spectroscopy (CD). The association constants between PAEs and HSA were determined using the Stern-Volmer and Scatchard equations. The binding of dimethyl phthalate (DMP) to HSA has a single class of binding site and its binding constants (K) are 4.08 × 10(3), 3.97 × 10(3), 3.45 × 10(3), and 3.20 × 10(3)L mol(-1) at 289, 296, 303, and 310K, respectively. The Stern-Volmer and Scatchard plots both had two regression curves for HSA-butylbenzyl phthalate (BBP) and HSA-di-2-ethylhexyl phthalate (DEHP), which indicated that these bindings were via two types of binding sites: the numbers of binding site for the first type were lower than for the second type. The binding constants of the first type binding site were higher than those of the second type binding site at corresponding temperatures, the results suggesting that the first type of binding site had high affinity and the second binding site involved other sites with lower binding affinity and selectivity. The thermodynamic parameters of the binding reactions (ΔG°, ΔH° and ΔS°) were measured, and they indicated the presences of hydrophobic forces and hydrogen interactions in the PAEs-HSA interactions, which agreed well with the results from molecular modelling. The alterations of protein secondary structure in the presence of PAEs were confirmed by UV-vis and CD spectroscopy. The time-resolved fluorescence study showed that the lifetime of Trp residue of HSA decreased after the addition of PAEs, which implied that the Trp residue of HSA was the main binding site.