The effect of calcium ions on the separation of scheelite from fluorite was studied using atomic force microscopy (AFM) measurement, X-ray photoelectron spectroscopy (XPS) analysis and micro-flotation experiment. The XPS results confirmed the adsorption of calcium on scheelite and fluorite surface, and adsorption difference existed between calcium solution and gypsum solution. According to the AFM results, the adhesion forces between scheelite and fluorite would increase in the presence of calcium, so did the corresponding jump-in distance and separation distance. Besides, jump-in distance and separation distance in calcium solution were larger than those in gypsum solution. When using sodium silicate as depressant, the adhesion forces between scheelite and fluorite decreased observably, while regained in the presence of calcium ions. Micro-flotation results confirmed the depression of calcium on scheelite and fluorite. Besides, both scheelite and fluorite were depressed strongly by sodium silicate in the presence of calcium. These indicated that the adsorption of CaOH+ would increase their hydrophilicity and enhance the interaction forces. Meanwhile, the selectivity of sodium silicate was depressed in the presence of calcium ions. The combined results could help to understand how calcium-mineral surface properties would cause the difficult separation.