Using various chromatographic experiments, six asterosaponins ( 1 − 6 ) were isolated from the MeOH extract of the Vietnamese starfish Astropecten monacanthus . The cytotoxic activities of the MeOH extract and six asterosaponins were evaluated on three human cancer cell lines, HL-60 (promyelocytic leukemia), PC-3 (prostate cancer), and SNU-C5 (colorectal cancer). Relative to the effects of the postitive control mitoxantrone, the MeOH extract (with IC₅₀ values ranging from 0.84±0.03 to 3.96±0.14 µg/mL) and astrosterioside D ( 5 ) (with IC₅₀ values ranging from 4.31±0.07 to 5.21±0.15 µ M ) exhibited potent cytotoxic effects against all three tested human cancer cell lines. In addition, the MeOH extract and astrosterioside D ( 5 ) have an effect on leading to apoptosis. Interestingly, the apoptosis of induction was accompanied by down-regulation of phosphatidyl inositol 3-kinase (PI3K)/AKT signaling and extracellular signal-regulated kinase (ERK) 1/2 mitogen-activated protein kinase (MAPK) signaling, and decrease of c-myc expression. Further studies are required to establish use of the asterosaponins from A. monacanthus as remedial and/or nutraceutical purposes.