In this paper, the effect of depressant carboxymethyl cellulose (CMC) on the separation of scheelite from calcite was studied. Micro-flotation experiments results indicated that CMC could significantly depress the floatability of calcite, but its adverse effect on the floatability of scheelite was very small. The flotation recovery of calcite decreased from 90% to 17% and the flotation separation of scheelite from calcite can be achieved at pH 9.5 when the concentration of CMC reached 2.0 mg/L. Zeta potential tests results revealed that the adsorption of -RCOO-species of CMC on the opposite positively charged calcite surface was more intense than scheelite. The FTIR results showed that CMC was chemisorbed on both scheelite and calcite surfaces, but the adsorption on calcite surface was more intensive. The XPS results depth revealed that the relative content of carbon was increased by 3.4% on calcite surface, which was greater than that of scheelite by 0.62%. And the shifts of binding energy on calcite surface were more obvious than scheelite. Atomic force microscopy (AFM) imaging intuitively showed the adsorption density and coverage of CMC on calcite surface was higher than that of scheelite. Finally, an adsorption model of regents on mineral surfaces could be obtained.