Pyrite is a major gangue mineral associated with galena and other valuable minerals, and it is necessary to selectively remove pyrite to upgrade the lead concentrate by froth flotation. In this study, the flotation experiments of a single mineral and mixed minerals were performed using chitosan with different molecular weights (MW = 2−3, 3−6, 10 and 100 kDa) as a depressant, ethyl xanthate as a collector, and terpineol as a frother, in a bid to testify the separation of pyrite from galena. Flotation results showed that the selective flotation of pyrite from galena can be achieved under the preferred reagent scheme, i.e., 400 g/t chitosan (10 kDa), 1600 g/t ethyl xanthate, and 100 g/t terpineol, while chitosan with other molecular weights cannot. Furthermore, the results of the zeta potential and contact angle measurements revealed that chitosan (10 kDa) has a strong adsorption on galena yet a very weak adsorption on pyrite at the dosage of 400 g/t. This study showed that chitosan (10 kDa) has great potential in the industrial flotation separation of pyrite from lead concentrates.