In this work, 2-phosphonobutane-1,2,4-tricarboxylic acid (PBTCA) was explored as a novel sphalerite depressant in the selective flotation of galena from sphalerite. The separation performances were investigated by single and synthetic flotation tests. The depression mechanism was examined by Fourier Transform Infrared spectroscopy (FTIR), zeta potential and X-ray Photoelectron Spectroscopy (XPS) analysis. The flotation test results showed that PBTCA was an effective sphalerite depressant with good selectivity at broad pH range, which could achieve satisfactory separation results. Zeta potential and FTIR results indicated that PBTCA was more prone to adsorb on sphalerite surface than that of galena. In addition, SIBX could replace adsorbed PBTCA on the galena surface while it was impossible to substitute that on the sphalerite surface, which rendered sphalerite surface hydrophilic and enlarged the floatability difference. XPS analysis manifested that PBTCA was adsorbed on sphalerite surface through the strong complexation between carboxyl, phosphonic acid groups and Zn sites on sphalerite surface, forming C-O-Zn or P-O-Zn bond.