Neonicotinoids, which have a wide range of applications, are systemic insecticides that efficiently control several sucking pests. Recently, thiamethoxam (THIA) has become one of the popular pest control agents. In vivo study, the toxicity of THIA was evaluated against a laboratory strain of cowpea aphid (Aphis craccivora Koch.) using the leaf dipping method for 24 and 48 hrs of exposure. In silico study, THIA was docked to acetylcholine binding protein (AChBP) to explain their binding mode of interaction. THIA exhibited good toxicity against adult aphids with LC₅₀ of 2.44 ppm at 24 hrs post-treatment, which greatly decreased to 0.75 ppm at 48 hrs post-treatment. THIA showed better binding interaction with AChBP than the co-crystalized ligand (imidacloprid) and good alignment in the active site. THIA indicated different variations in the activities of tested enzymes. Where, aphids treated with LC₅₀ of THIA increased the biochemical activities of acetylcholinesterase, antioxidant enzymes and peroxidases while decreasing the activity of alpha esterase, carboxylesterase (CarE), glutathione S-transferase and catalase. Therefore, this compound is efficient against sap-sucking pests with good irreversible and specific binding interaction with nicotinic acetylcholine receptor (nAChR) and, consequently, low mammalian toxicity. So, THIA is considered a favourable agent for the management of insect pests.