Autophagy deficiency in macrophages exacerbates inflammation in atherosclerosis (AS), and recently, galectin-3 (Gal-3) has been implicated as a critical promoter of inflammation in AS. Further, melatonin (Mel) exerts an autophagy-promoting effect in many chronic inflammatory diseases. In this study, we aimed to investigate whether Mel inhibits AS progression by downregulating Gal-3 to enhance autophagy and inhibit inflammation. Thus, we performed in vivo and in vitro experiments using high-fat diet (HFD)-fed ApoE  mice and THP-1 macrophages, respectively. Smart-seq of AS plaque macrophages revealed that the differentially expressed genes (DEGs) downregulated by Mel were enriched in immune-related processes, and changes in inflammation status were confirmed based on lower levels of proinflammatory factors in Mel-treated HFD-fed ApoE  mice and THP-1 macrophages. Further, via transcriptome-based multiscale network pharmacology platform (TMNP), the upstream target genes of the smart-seq DEGs were identified, and Gal-3 showed a high score. Gal-3 was downregulated both in vivo and in vitro by Mel treatment. Besides, the enrichment of the target genes predicted via the TMNP method indicated that autophagy considerably affected the DEGs. Mel treatment as well as Gal-3 knockdown downregulated most inflammatory response-related proteins could attribute to enhancing autophagy. Mechanistically, Mel treatment inhibited Gal-3 leading to lowering the activity of the nuclear transcription factor-kappa B (NF-κB) pathway, and promoting the nuclear localization of transcription factor EB (TFEB). However, increased secretion of Gal-3 activated the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) pathway and impaired autophagy via binding to CD98. Thus, Mel promoted autophagy and restrained inflammation by downregulating Gal-3, implying that it holds promise as a treatment for AS.