Chemical investigation of the EtOAc extract from the aerial parts of Acrostichum aureumdistributed along coastlines of Vietnam lead to the isolation of a new ecdysteroid, named pterosterone 20,22-acetonide (1), along with twelve known compounds (2-13). Their structures were elucidated by spectroscopic methods including 1D, 2D NMR and HR-ESI-MS analysis as well as comparison with the results of previous studies. Five ecdysteroids (1-5) were evaluated for their cytotoxic activity against the LU-1, MCF7 and HepG2 human cancer cell lines using the SRB assay. Ecdysteroids 1 and 2 having a 20,22-dimethyl acetal group showed cytotoxicity against all tested cell lines with IC50 values in the range 51.59 to 60.14μM. The other compounds were considered as inactive in this test.