A novel petrosiacetylene analog (petrosiacetylene E) has been isolated from the Korean marine sponge Petrosia sp., along with petrosiacetylene A, B and C. Their structures were elucidated on the basis of spectroscopic methods and the stereochemistry of the new compound was determined by using the modified Mosher's method. Petrosiacetylene E showed higher cytotoxicity against five human cancer cell lines than petrosiacetylene A and B, presumably due to the additional hydroxy group located at C-16.