The rhizome of Dioscorea birmanica Prain & Burkill was commonly used by Thai folk doctors to treat cancer patients. The previous report was found that its extract exhibit high cytotoxic against lung cancer cells. Thus, the objective of this research was to investigate the cytotoxic activity against lung cancer cells using Dioscorea birmanica Prain & Burkill extract and an isolated cytotoxic compound therein. The ethanolic extract of Dioscorea birmanica Prain & Burkill showed cytotoxic activity against two types of lung cancer cell lines (CORL23 and A549) and against normal lung cells (MRC5) (IC50=8.7, 7.5 and 94.8µg/ml respectively) in the sulphorhodamine B assay [1,2]. Bioassay guided fractionation was used to isolate the cytotoxic saponin diosgenin-3-O-α-l-rhamnosyl (1 → 2)-β-d-glucopyranoside or Prosapogenin A of dioscin (DBS1). DBS1 showed high cytotoxic activity against two types of lung cancer cells; CORL23 and A 549 (IC50=1.8., 1.8µg/ml respectively) but less cytotoxic activity against normal lung cells MRC5 (IC50=37.1µg/ml). These results can support using this plant to treat cancer by Thai folk doctors.