Abstract: Developing green, eco-friendly and high-performance depressants is one of the key strategies to achieve efficient flotation separation of fluorite from calcite. In this study, grape seed extract (GSE) was introduced into the fluorite-calcite flotation system, and its effect on the flotation separation of the two minerals was systematically investigated. Micro-flotation test results show that with 30 mg·L–1 sodium oleate (NaOL) as a collector and at pH 8, the addition of 5 mg·L–1 GSE increased the fluorite grade and recovery of the concentrate by 21.61wt% and 3.44wt%, respectively. Contact angle measurements, NaOL adsorption capacity detections and Zeta potential determination revealed the underlying mechanism at the macroscopic level: GSE has little effect on the adsorption of NaOL on fluorite surfaces, but preferentially adsorbs onto calcite surfaces, thereby effectively depressing NaOL adsorption on calcite. Fourier transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS) and density functional theory (DFT) simulation further verified at the molecular-atomic level that GSE molecules formed multidentate chelating coordination with Ca2+ on calcite surfaces via oxygen atoms in phenolic hydroxyl groups, clarifying the microscopic pathway for its selective depression of calcite. This study provides a green scheme for fluorite-calcite flotation, reveals GSE’s calcite depression mechanism, and guides fluorite recovery.