Trimethyltin chloride (TMT) is a potent neurotoxin that causes neuroinflammation and neuronal cell death. Melatonin is a well‐known anti‐inflammatory agent with significant neuroprotective activity. Male C57BL/6J mice were intraperitoneally injected with a single dose of melatonin (10 mg/kg) before exposure to TMT (2.8 mg/kg, ip). Thereafter, the mice received melatonin (10 mg/kg, ip) once a day for another three consecutive days. Melatonin dramatically alleviated TMT‐induced neurotoxicity in mice by attenuating hippocampal neuron loss, inhibiting epilepsy‐like seizures, and ameliorating memory deficits. Moreover, melatonin markedly suppressed TMT‐induced neuroinflammatory responses and astrocyte activation, as shown by a decrease in inflammatory cytokine production as well as the downregulation of neurotoxic reactive astrocyte phenotype markers. Mechanistically, serine peptidase inhibitor clade A member 3N (SERPINA3N) was identified as playing a central role in the protective effects of melatonin based on quantitative proteome and bioinformatics analysis. Most importantly, melatonin significantly suppressed TMT‐induced SERPINA3N upregulation at both the mRNA and protein levels. The overexpression of Serpina3n in the mouse hippocampus abolished the protective effects of melatonin on TMT‐induced neuroinflammation and neurotoxicity. Melatonin protected cells against TMT‐induced neurotoxicity by inhibiting SERPINA3N‐mediated neuroinflammation. Melatonin may be a promising and practical agent for reducing TMT‐induced neurotoxicity in clinical practice.