In this research, a novel collector cetyl trimethyl ammonium chloride (CTAC) was used to separate hematite from quartz via reverse flotation for the first time. Micro-flotation tests showed that CTAC had a strong ability to selectively collect quartz and a separation of hematite from quartz could be accomplished with a concentration of 0.00263 mmol/L CTAC. Zeta-potential measurements indicated that the positive CTAC+ species could selectively increase the surface potential of quartz, but it had rather a weak effect on the hematite surface. X-ray photoelectron spectroscopy (XPS) detection indicated that CTAC had a stronger binding affinity to oxygen sites on the surface of quartz than that of hematite, resulting in a large amount of CTAC predominantly adsorbed on quartz rather than hematite.