This study aims to identify the underlying mechanism by which melatonin protects neurons. Firstly, the inhibitory effect of melatonin on ferroptosis was verified by treating HT22 cells with melatonin, Erastin, and Ferrostatin-1. Secondly, transcriptomic and metabolomic analyses were performed. Melatonin-related hub genes were identified by differential gene expression analysis, and lipid metabolism-related critical signaling pathways and biological processes (BPs) were determined by gene set enrichment analysis (GSEA). Finally, the expression of hub genes was verified by quantitative real-time PCR (qRT-PCR) or Western Blot (WB), and the involvement of Tribble 3 (Trib3) in the regulation of lipid metabolism and ferroptosis by melatonin was confirmed by Cell Counting Kit 8 (CCK-8) assay, ROS analysis, and WB. Assay results showed that melatonin significantly increased Gpx4 activity, decreased ROS generation, and inhibited ferroptosis in HT22 cells. The hub gene Trib3 was obtained by transcriptomic analysis, and its expression was upregulated with Erastin treatment. Lipid metabolomic analysis suggested that the regulation of lipid metabolism by melatonin was associated with glycerophospholipids. In vitro experiments showed that Trib3 was regulated by the upstream factor Atf4, and the protein levels of Trib3 and Atf4 were significantly increased after Erastin treatment. However, melatonin can reduce the protein levels of Trib3 and Atf4, increase the survival rate of HT-22 cells and the activity of GPX4, and reduce the ROS content. Melatonin inhibits neuronal ferroptosis by affecting the Atf4/Trib3 axis via the modulation of lipid metabolism.