Since the introduction of imidacloprid in the early 1990s, it has become one of the most widely applied insecticides, and currently represents about 20% of the global pesticide market (Tomizawa, M.; Casida, J. E. J. Agric. Food Chem 2011, 59, 2883-2886). In the context of this study, our major aim was to comprehensively scrutinize the nature of imidacloprid with two typical model proteins, lysozyme and albumin, by means of circular dichroism (CD), steady-state and time-resolved fluorescence, and molecular modeling at the molecular level. Far-UV CD verified that the spatial structure of both proteins was altered with a distinct reduction of α helix in the presence of imidacloprid suggesting unfolding of the protein (i.e., protein damage). The data of steady-state and time-resolved fluorescence showed that the conjugation of imidacloprid with lysozyme yielded quenching by a static mechanism (KSV = 3.841 × 10(4) M(-1)), while combined static and dynamic properties existed for albumin tryptophan (Trp)-214 fluorescence. Molecular modeling simulations displayed that the imidacloprid binding site was near to the Trp-62 and Trp-63 residues of lysozyme, and it was located at the subdomain IIA (warfarin-azapropazone site) of albumin. Furthermore, the primary forces between protein and imidacloprid are hydrogen bond, hydrophobic, and π-π interactions, but the affinity of lysozyme with imidacloprid is much lower than albumin, probably because the affinity distinctions stem from discrepancy in the three-dimensional structure of the two globular proteins. The results presented here will help to further understand the credible mechanism by which the toxicological implication of neonicotinoid insecticides is palliated by carrier protein.