We observed that (1S,2S,3E,7E,11E)-3,7,11,15-Cembratetraen-17,2-olide (LS-1), marine cembrenolide diterpene, inhibited growth and induced apoptosis in colon cancer cells via a reactive oxygen species (ROS) dependent mechanism. Treatment of HT-29 cells with LS-1 resulted in ROS generation, which was accompanied by disruption of mitochondrial membrane potential, cytosolic release of cytochrome c, sub-G1 peak accumulation, activation of Bid, caspase-3, -8, and -9, and cleavage of poly(ADP-ribose) polymerase (PARP) along with the suppressive expression of B cell lymphoma-2 (Bcl-2). All these effects were significantly blocked on pretreatment with the ROS inhibitor N-acetylcysteine (NAC), indicating the involvement of increased ROS in the proapoptotic activity of LS-1. Moreover, we showed that LS-1 induced the phosphorylation of c-Jun N-terminal kinase (JNK) and dephosphorylation of p38, extracellular signal-regulated kinase (ERK), Akt, Src and signal transducer and activator of transcription (STAT)3, which were effectively attenuated by NAC. In addition, the expressions of antioxidant catalase and glutathione peroxidase were abrogated by treatment using LS-1 with or without NAC. These findings reveal the novel anticancer efficacy of LS-1 mediated by the induction of apoptosis via ROS generation in human colon cancer cells.