Addressing the persistent challenge of separating smithsonite from calcite using flotation method, this study explores the impact of carboxymethyl chitosan (CMCS) on selective separation using sodium oleate (NaOL) assisted flotation. Results indicated a substantial reduction in calcite recovery to 2.25 % with the addition of 40 mg/L CMCS at pH 9, while recovery of smithsonite remained essentially unchanged at 94.78 %. Moreover, we found that using CMCS as the depressant can effectively separate smithsonite from calcite, based on the flotation testing results with artificially mixed minerals. Contact angle tests results showed that CMCS can significantly lower the surface hydrophobicity of calcite without any negative effect on that of smithsonite when using NaOL as a collector. TOC, FTIR, AFM, and ToF-SIMS analyses demonstrated stronger adsorption of CMCS on the surface of calcite compared to smithsonite. XPS data, solution chemical analysis and DFT revealed interaction between –COO- in CMCS with Ca sites on the surface of calcite because of the electrostatic adsorption and chemical adsorption, forming −COOCa. It leaded to shielding effects on the NaOL adsorption stemming, which makes NaOL more adsorbed on smithsonite surface.