The effect of hydroxypropyl starch (HPS) on the flotation separation of fluorite from calcite using sodium oleate (NaOl) as a collector was studied. Micro-flotation results of single minerals showed that both fluorite and calcite presented good floatability in the presence of NaOl. However, by adding HPS before NaOl, the calcite flotation recovery was selectively reduced. The flotation of artificially mixed minerals confirmed that an efficient separation of fluorite from calcite could be achieved using the reagents scheme of HPS/NaOl. Zeta potential measurements demonstrated that HPS prevented the NaOl adsorption onto the calcite surface while it did not inhibit the interaction of NaOl with the fluorite surface. Fourier Transform Infrared (FT-IR) analysis was also revealed that the selective adsorption of HPS onto the calcite surface.