Witherite has been widely used as an industrial and environmental source of barium, with calcite being the primary associated carbonate mineral. However, few studies have been conducted to effectively concentrate witherite from barium ores. In this work, with the treatment of potassium chromate (K2CrO4) and sodium oleate (NaOL), witherite was selectively separated from calcite through selective flotation at different pH conditions. In addition, contact angle, Zeta potential, adsorption and X-ray photoelectron spectroscopy measurements were performed to characterize the separation mechanisms. The results demonstrated that NaOL had a strong collecting ability for both witherite and calcite; nevertheless, witherite could be effectively selected from calcite with the highest recovery at pH 9 in the presence of K2CrO4. From the XPS measurements, NaOL and K2CrO4 were found to be primarily attached to the surfaces of witherite and calcite through chemisorption. The presence of K2CrO4 on the surface of calcite adversely influenced the NaOL adsorption, which could make the flotation separation efficient and successful.