Abstract: Neonicotinoid insecticides cycloxaprid have potential harm to non-target organisms. However, there is a lack of research on its toxicological mechanisms, in vivo transport mechanisms, and other aspects. In this study, the binding mode of cycloxaprid with human serum albumin (HSA) was investigated using a combination of the following test methods: fluorescence quenching, molecular docking, molecular dynamics, binding free energy calculation, and alanine scanning. The results indicated that: 1) Cycloxaprid can effectively quench HSA, and their binding constants range from 0.76 × 10⁵ L/mol to 1.57 × 10⁵ L/mol at different temperatures with strong binding ability. 2) Cycloxaprid binds to the IIA hydrophobic cavity of HSA with one binding site. 3) By combining free energy analysis and thermodynamic parameter calculation, the results showed that the main binding forces between cycloxaprid and HSA are van der Waals forces and hydrogen bonding interactions. 4) By molecular dynamics simulation, the results showed that the binding free energy is ‒25.90 kJ/mol, which formed a relatively stable complex. 5) The alanine mutation scan results showed that the amino acid residue Gln459 is a key amino acid that binds to cycloxaprid and HSA. This research can provide a theoretical basis for elucidating the toxicology mechanism of epoxidizine in the human body.