The separation of molybdenum and iron is one of key issues of the hydrometallurgical preparation of pure molybdenum chemical products. The thermodynamic equilibrium diagrams for distribution of species at different pH values and different concentrations of molybdenum, iron and sulfur with the systems of Mo(VI)-H2O, Fe(III)-S(VI)-H2O and Mo(VI)-Fe(III)-S(VI)-H2O at 298 K were established, separately. Thermodynamic analysis results revealed that both molybdenum and iron were transformed from their anions to cations with the decrease in pH values. The pHMf0.5 for the conversion of molybdenum anions to cations decreased from 0.92 to 0.20 with the increase in the molybdenum concentration from 0.05 mol/L to 1 mol/L (pHMf50 is defined as the pH value when target metal anions occupy 50% mole fraction of total). The total sulfur concentration has significant effect on the conversion of iron (III) species with the pH change. The separation of molybdenum (VI) and iron (III) from acidic solutions using a tertiary amine extractant N235 could be achieved by decreasing the total sulfur concentration and controlling the equilibrium pH in an optimum range. The verification test results showed that the operating window for the separation of Mo(VI) and Fe(III) from a solution containing 0.01 mol/L [Mo]T, 0.05 mol/L [Fe]T and 1.15 mol/L [S]T was in the pH range of 0.5–1.0, which was consistent with the thermodynamic analysis.