The issue that eliminating the negative effects of inevitable ions in the pulp on mineral flotation separation has attracted widespread attention. To achieve the efficient flotation separation of limonite from quartz, citric acid was utilized as a depressant for quartz in this paper. The solubility of limonite was studied using inductively coupled plasma tests (ICP), and the effect of Fe3+ and citric acid on the separation of limonite from quartz under sodium oleate (NaOL) system was investigated using batch flotation tests. The underlying mechanisms were probed through zeta potential measurements, infrared spectroscopy (IR), X-ray photoelectron spectroscopy (XPS), and contact angle detection. It was found that limonite could dissolve a certain number of Fe3+ in the pulp. These Fe3+ adsorbed onto quartz and acted as the active sites to promote the adsorption of NaOL, thereby enhancing quartz flotation evidently and deteriorating the separation of limonite from quartz. Fortunately, citric acid could depress the flotation of iron-activated quartz quite well by covering and desorbing the active sites of Fe3+ and causing the steric hindrance to prohibit the adsorption of NaOL. Meanwhile, the hydrophilic groups in citric acid could also increase the surface hydrophilicity of quartz and enhance the inhibitory effect. In comparison, Fe3+ and citric acid had little effect on the adsorption of NaOL onto limonite since its surface exposed enough Fe-active sites for the adsorption of NaOL. Thus, the difference in the floatability of limonite and quartz was enlarged, eventually achieving a better separation of them. This research sheds new light on the role of citric acid in the selective separation of quartz from soluble iron minerals.