OBJECTIVE To explore the effects of ANO1 overexpression on the proliferation, detachment, spreading, and migration of laryngocarcinoma Hep-2 cell line. METHODS ANO1-overexpressing Hep-2 cell line was selected as the assay group, and Hep-2 cell line with empty plasmid was selected as the control group. MTT assay was used to detect the proliferation abilities of Hep-2 cells in both two groups. Cell detachment assay and spreading assay were used to detect the detachment and spreading abilities of Hep-2 cells. Boyden chamber invasion assay, wound healing assay in vitro, and niflumic acid block chloride channel were used to detect the migration abilities of Hep-2 cells. All data were analyzed by SPSS 10.0 software package. RESULTS Cell proliferation assay by MTT showed that, compared with the control group, the optical density value of assay group was not significantly different (P=0.62). The results of cell detachment assay and cell spreading assay showed the cell detachment rates and cell spreading rates in assay group were significantly higher than those in control group (P<0.0001). The results of Boyden chamber invasion assay showed the percentages of cells migrating through the membrane in assay group were significantly higher than those in control group (P<0.0001). The results of in vitro wound healing experiments showed the wound area rate in assay group was significantly lower than that in control group (P<0.0001). The results of niflumic acid blocking chloride channel experiments showed the wound area rates in assay group were significantly higher than those in control group (P<0.0001). CONCLUSION ANO1 overexpression does not remarkably alter the proliferation rate of cancer cells, but increases the migration, spreading, and detachment capacities of head and neck squamous cell carcinoma.