The influences of long-term static interaction between flotation reagents and minerals on mineral surface properties and their flotation behaviors were investigated in this work. Single mineral results showed that when the static interaction time between flotation reagents and minerals increased from 0 to 8 days, there were no significant differences in the recovery of fluorite (ranging from 77.50% to 74.50%), while the recovery of calcite dramatically decreased from 97.17% to 41.13%. The collector adsorption results indicated that after the long-term static interaction between the collector and minerals, adsorption and desorption of collector on the minerals occurred, and the adsorption amount of collector on fluorite and calcite varied from 0.396 mg/g to 0.421 mg/g, and from 0.444 mg/g to 0.404 mg/g, respectively. The contact angles of fluorite and calcite decreased from 134.3° and 105.0° for 0 days to 109.7° and 52.5° for 8 days, respectively, which demonstrated that the fluorite had a higher hydrophobicity than that of calcite after 8 days of static interaction between the minerals and collector. The Fourier transform infrared spectroscopy (FTIR) analysis showed that there were chemisorptions between the collector and the surfaces of the fluorite and calcite. Meanwhile, the X-ray photoelectron spectroscopy (XPS) results further elucidated that the amount of C=O from the oleic acid (OA) molecule for calcite treated for 8 days was less than for that treated for 0 days. In addition, the flotation results of the tailings from Jiangxi containing fluorite and calcite, indicated that the grade and recovery of fluorite increased from 87.18% and 40.99% for 0 days to 93.00% and 46.01% for 8 days, respectively, indicating that the separation of fluorite from calcite could be achieved via increasing the long-term static interaction between the flotation reagents and minerals.