Pyrite weathering often occurs in nature and causes heavy metal ion pollution and acid mine drainage during the process. Humic acid (HA) is a critical natural organic material that can bind metal ions, thus affecting metal transfer and transformation. In this work, in situ electrochemical techniques combined with spectroscopic analysis were adopted to investigate the interfacial processes involved in pyrite weathering with/without HA. The results showed that the pyrite weathering mechanism with/without HA is FeS2 → Fe2+ + 2S0 + 2e-. The presence of HA did not change the pyrite weathering mechanism, but HA adsorbs on the pyrite surface and inhibits the further transformation of sulfur. Furthermore, HA and Fe(II) ions can form complex at 45.0 °C. Increased concentration of HA, decreased HA solution acidity or decreased environmental temperature would all weaken the pyrite weathering, for the above conditions cause pyrite weathering to have a larger resistance of the double layer and a larger passive film resistance. Pyrite will release 73.7 g m-2·y-1 Fe2+ to solution at pH 4.5, and the amount decreases to 36.8 g m-2·y-1 in the presence of 100 mg/L HA. This study provides an in situ electrochemical method for the assessment of pyrite weathering.