The application of guar gum for pyrite depression in seawater flotation was assessed through microflotation tests, Focused Beam Reflectance Measurements (FBRM), and Particle Vision Measurements (PVM). Potassium amyl xanthate (PAX) and methyl isobutyl carbinol (MIBC) were used as collector and frother, respectively. Chemical species on the pyrite surface were characterized by Fourier-transform infrared spectroscopy (FTIR) spectroscopy. The microflotation tests were performed at pH 8, which is the pH at the copper sulfide processing plants that operate with seawater. Pyrite flotation recovery was correlated with FBRM and PVM characterization to delineate the pyrite depression mechanisms by the guar gum. The high flotation recovery of pyrite with PAX was significantly lowered by guar gum, indicating that this polysaccharide could be used as an effective depressant in flotation with sea water. FTIR analysis showed that PAX and guar gum co-adsorbed on the pyrite surface, but the highly hydrophilic nature of the guar gum embedded the hydrophobicity due to the PAX. FBRM and PVM revealed that the guar gum promoted the formation of flocs whose size depended on the addition of guar gum and PAX. It is proposed that the highest pyrite depression occurred not only because of the hydrophilicity induced by the guar gum, but also due to the formation of large flocs, which could not be transported by the bubbles to the froth phase. Furthermore, it is shown that an overdose of guar gum hindered the depression effect due to redispersion of the flocs.