Introduction: Vascular calcification (VC) is a common pathological feature in chronic kidney disease, atherosclerosis, and aging. Nobiletin (NOB) is a natural compound which has various biological activities. This study aimed to explore the effect of NOB on VC and its molecular mechanism. Methods: Primary vascular smooth muscle cells (VSMCs) were cultured in calcification medium for 3 days to induce calcification. For NOB-treated groups, 10µM NOB was added into medium or calcification medium. A mouse model of VC was established by intraperitoneal injection of vitamin D, and mice were orally gavaged with 50mg/kg/day NOB or vehicle. Alizarin-Red staining and calcium content quantitation were used to visualize calcium deposition. Western blot analysis, co-immunoprecipitation, quantitative proteomic analyses and methyltransferase assays were performed to detect protein expression, protein–protein interactions, and post-translational modification levels. Results: Network pharmacology analysis identified NOB as a potential therapeutic agent for VC. In vitro, Alizarin-Red staining, calcium content quantitation, qRT-PCR and Western blot showed that NOB prevented calcium accumulation in VSMCs and downregulated osteogenic markers (RUNX2, OPN, BMP2). In the vitamin D-induced mouse aortic calcification model, NOB inhibited calcium deposition without affecting serum calcium levels and decreased osteogenic marker expression in aortas. Quantitative proteomic analyses suggested that NOB-induced upregulation of deleted in liver cancer-1 (DLC1) might be a key factor in inhibiting VC. DLC1 overexpression reduced calcium deposition and osteogenic marker expression in VSMCs, while knockdown of DLC1 aggravated calcification. Mechanistically, NOB upregulated RAR-related orphan receptor alpha (RORα) expression, which competed with DLC1 for binding to enhancer of Zeste homolog 2 (EZH2). This competitive interaction inhibited EZH2-mediated methylation and degradation of DLC1, preserving its function in VSMCs. Conclusion: Our findings highlight a novel therapeutic mechanism of NOB in vascular calcification through modulation of the RORα–EZH2–DLC1 axis, providing mechanistic insights into natural-compound-mediated regulation of vascular pathology.