Cancer stem cells (CSCs) have the ability to dictate tumor initiation, recurrence, and metastasis. Here, we examined the expression of aα2δ1+ in laryngeal cancer tissues and further determined the effect of α2δ1 on the migratory ability and tumorigenicity of laryngeal cancer cells. Immunofluorescence staining revealed that α2δ1 was positive in 13 (13/16, 81.25%) cases in laryngeal squamous cell carcinoma (LSCC) tissues, 7 (7/16, 43.75%) cases in paracancerous tissues and only 2 (2/16, 12.5%) cases in normal tumor tissues. Our quantitative RT-PCR assays further showed that α2δ1+ LSCC cells expressed significantly higher levels of stem cell-associated genes and drug efflux and resistance genes versusα2δ1- cells. Sphere-forming assays demonstrated higher sphere-forming efficiency in the α2δ1+versusα2δ1- subpopulation. Our Matrigel assays showed that α2δ1+ cells exhibited significantly greater invasive and migratory ability than α2δ1- cells. Furthermore, the percentage of purified α2δ1+ in TU686 and TU212 cells treated cisplatin or paclitaxel was significantly higher than that of the control group. Tumor xenograft assays revealed that the tumorigenicity of α2δ1+ cells was much higher than α2δ1- cells. In conclusion, a α2δ1+ subpopulation with CSC-like property was present in laryngeal cancer and possessed high self-renewal activity and was sufficient for tumor growth, differentiation, migration, invasion, and chemotherapeutic resistance. They could represent a promising therapeutic target for LSCC.