The objective of this study was to analyze the antiproliferative activity of Dihydrosinularin, a cembranolide isolated from the soft coral Sinularia flexibilis against cancer CL1-5 cells and to determine the mode of cancer cytotoxicity. An Thiazolyl Blue Tetrazolium Blue (MTT) assay was used to determine the cytotoxic effect of dihydrosinularin. Subsequently, apoptosis and cell cycle analyses were performed by using flow cytometry. Apoptosis, G2/M cell cycle control and DNA-damage response protein expression were assessed using Western blotting. Using the MTT method, we discovered that dihydrosinularin dramatically reduced growth of CL1-5 cells. By using a flow cytometry assay, it was demonstrated that dihydrosinularin caused G2/M cell cycle arrest and dependent apoptosis in CL1-5 cells. JC-1 fluorescence labeling was also used to show the reduction of mitochondrial membrane potential (MMP) in dihydrosinularin-treated cells. In addition, dihydrosinularin influenced the expression levels of apoptotic and G2/M cell cycle arrest-related proteins. Moreover, the expression of -H2AX considerably, showing that dihydrosinularin caused DNA double-strand breaks. The ATM/Chk2 DNA damagee response signaling pathway was then activated. This study showed that dihydrosinularin promoted apoptosis, G2/M arrest, and DNA damage pathways, which could be important for cytotoxicity in human non-small cell lung cancer cells.