Four new water‐soluble polysaccharides has previously been isolated from Cardamine hupingshanensis. In this experiment, the structure of C. hupingshanensis polysaccharide NO. 3 (CHP‐3) is analyzed and its in vitro anti‐tumor activity is assessed. Through structural analysis, CHP‐3 is found to have a molecular weight of 80.64 kDa, and be mainly composed of glucose, galactose, and L‐arabinose; The backbone of CHP‐3 is mainly composed of →5)‐α‐L‐Araf‐(1→ and →4)‐α‐D‐Glc(p)‐(1→), respectively. By studying its anti‐tumor activity in vitro, it can be concluded that treatment with different concentrations of CHP‐3 exerted diverse effects on A549 cells, with an obvious dose‐dependent relationship. Moreover, after treatment with 12.8 mg mL−1 CHP‐3, significant inhibitory effects are observed on the proliferation, migration, and invasion of A549 cells, indicating that it can be used as an anti‐tumor agent for continued in‐depth research.