In this paper, the effect of pyrite on the activation of sphalerite was investigated by micro-flotation, copper adsorption experiments, X-ray photoelectron spectroscopy (XPS), and electrochemical measurement. The micro-flotation test results showed that the recovery and flotation rate of sphalerite with copper sulphate as activator and butyl xanthate as collector were significantly decreased with the increasing content of pyrite in pulp. Cu²⁺ adsorption results indicated that the adsorption of Cu²⁺ on the sphalerite surface were decreased when pyrite was present in the pulp. XPS surface analysis demonstrated that the proportion of Cu⁺ species increased in the activation products on the sphalerite surface, but the total atomic concentration of Cu atom was decreased. Linear voltammetry measurement suggested that the current density of Cu⁺ species oxidizing to Cu²⁺ species was increased when sphalerite was electrically contacted with pyrite, which confirmed the increased proportion of Cu⁺ species on Cu-activation sphalerite surface when contacting with pyrite. These results indicated that there is not only a competitive adsorption for cupric ions (Cu²⁺), but the galvanic interaction between sphalerite and pyrite also has a significant influence on the copper activation of sphalerite.