Sea cucumber is known as the source of bioactive secondary metabolites. Actinopyga is one of the sea cucumbers that have not been explored for its bioactivity. The objectives of this research were to assess the cytotoxic activity and to examine the characteristic of sea cucumber Actinopyga sp. methanolic extract. Cytotoxicity assay was conducted by using MTT method against W iDr (colon cancer) and T47D (breast cancer) cell lines. Actinopyga sp. methanolic extract was characterized by using phytochemical screening, fourier transform-infra red spectroscopy (FT-IR), and Liquid Chromatography Ion Trap Time of Flight Mass Spectrophotometer (LC-IT-ToF-MS). Results showed that Actinopyga sp. methanolic extract inhibit W iDr and T47D cell lines viability with the LC 50 value of 55.93 and 87.55 µg/ ml, respectively. Functional groups analysis showed the presence of hydroxyl, amine, carboxylic acid, nitrate, amide, sulphur, ester, and ether. The spectra mass analysis of crude extract showed that it contains steroid compounds.