Once diagnosed, esophageal cancer has a very low overall 5- year survival rate. This study investigates the mechanisms behind the invasiveness and severity of esophageal squamous cell carcinoma (ESCC). Materials and Methods: Transwell invasion chamber was used to subdivide one Taiwanese ESCC cell line, CE81T/VGH, into sublines (CE81T-0, CE81T-1, CE81T -2, CE81 T-3, and CE81T-4) in four rounds of assays; the most invasive were identified, and various factors related to their invasiveness measured. Results: CE81T-1, CE81T-2, CE81 T-3 and CE81T-4 sublines were significantly more invasive than the parental cells (CE81T/VGH) and CE81T- 0 subtitle. CE 81T-1 and CE81T-4, the sublines we chose to study further, had significantly greater colony-forming ability (3.5- to 2. 7-fold) and wound migrating activity (1.95- to 2.6-fold) than the parental cells in vitro (p