The impact of sulfuric acid pretreatment on the surface property of oxidized pyrrhotite (OX-pyrrhotite) and its application in flotation desulphurization of magnetite concentrate were investigated in this study. OX-pyrrhotite using sodium butyl xanthate (BX) collector performed a poor floatability. After sulfuric acid pretreatment, OX-pyrrhotite exposed a new sulphide surface similar to raw pyrrhotite. XPS analysis indicated that the oxidized species such as sulphate were dissolved away from the OX-pyrrhotite surface. This conversion made the natural hydrophobicity and isoelectric point of OX-pyrrhotite proximity to pyrrhotite. Contact angle and zeta potential measurements showed that the sulfuric acid pretreatment increased the adsorption of BX collector and the hydrophobicity of OX-pyrrhotite surface. FTIR and XPS analyses inferred that there existed weak adsorption of BX collector on OX-pyrrhotite surfaces. After sulfuric acid pretreatment, the interaction between BX collector and OX-pyrrhotite surfaces was strengthened, generating dixanthogen species. Therefore, the sulfuric acid pretreatment improved the floatability of OX-pyrrhotite within a wide pH region. When sulfuric acid pretreatment was adopted, the flotation desulfurization could decrease the sulfur content of magnetite concentrate from 4.50% to 0.18%.