Analysis on suppression effect of depressant on naturally hydrophobic gangue minerals is of great importance for both industrial applications and theoretical research. In this work, the role of glucan in the flotation separation of pyrite and pyrophyllite was studied and the selective depression mechanism of glucan on pyrophyllite was discussed. The flotation tests indicate that pyrophyllite is a naturally hydrophobic mineral. Using glucan as a depressant can achieve flotation separation of pyrite from pyrophyllite at the natural pH of the pulp. This is attributed to greater adsorption and stronger depression effect of glucan on pyrophyllite than that of pyrite. According to Zeta-potential, Fourier transform infrared, X-ray photoelectron spectroscopy and molecular dynamics simulation results, glucan would selectively absorb on the pyrophyllite surface by chemical and physical adsorption between the -OH hydrophilic group in glucan molecule and the Al/Si atom on pyrophyllite surface. Al-O chemical complexation is considered as the main adsorption mode.