Diethylenetriaminepenta (methylene-phosphonic acid) pentasodium salt (DTPMPA), an eco-friendly reagent, was tried for the first time as a depressant for flotation separation of scheelite from calcite. Micro-flotation tests show that DTPMPA can selectively depress the floatability of calcite. In contrast, DTPMPA barely affects the flotation behavior of scheelite. Based on the selective depress effect, floatation separation of scheelite and calcite can be effectively achieved by using DTPMPA as depressant in artificially mixed minerals flotation tests. Based on a series of measurements, it found the surface of calcite was positively charged due to the existence of Ca ion site, which can be chelated with -PO₃H- functional group on the surface of DTPMPA. In the flotation process, DTPMPA can be chemically absorbed on the surface of calcite to inhibit further collector adsorption. On contrast, little DTPMPA was adsorbed on the surface of scheelite due to the spatial site resistance and electrostatic repulsion induced by surface WO₄²⁺. All in all, these results exhibit DTPMPA has excellent selective depression ability on calcite, which can be potentially applied in the actual scheelite flotation process.