The nature and mechanism of interaction between carboxymethyl cellulose (CMC) with cassiterite (and chlorite surfaces) and their effects on the flotation separation process of cassiterite (from chlorite) were investigated by micro-flotation tests, surface adsorption experiments, zeta potential measurements, solution chemical calculation, infrared spectroscopy, and X-ray photo-electron spectroscopy (XPS). The results from single mineral tests revealed that CMC exhibited good selective inhibition effects with cassiterites and chlorites. When the dosage was 12.5 mg/L at pH 8, cassiterite and chlorite recovery was 92.2% and 6.3%, respectively. The artificial mixed ore test revealed that the flotation separation effect was the best when the dosage of CMC was 6.5 mg/L. Cassiterite used during the studies was 75.1% pure. The recovery was 82.8%. The interaction between CMC and the cassiterite surface led to a shift in the zeta potential toward the negative direction. CMC was weakly adsorbed on the cassiterite surface. There was no significant impact on the subsequent collection of sodium oleate. The concentration of C atom increased post interaction, and the potential shifted toward the negative direction. Characteristic CMC peaks were observed at this point. Hydrogen bonds and weak chemisorption interactions between CMC and chlorite affected the interaction between sodium oleate and the chlorite surface. It also affected the flotation results. The cassiterite and chlorite were separated effectively.