We demonstrated that an extract from Pylaiella littoralis, collected from the Federate States of Micronesia (FSM), could inhibit the proliferation of tumor cells. P. littoralis extract (PLE) showed anti-proliferative activities in the tumorigenic cells tested, ranging from 20.2% to 67.9%. The highest inhibitory activity, in HT-29 cells, was selected for further experiments. PLE showed no cytotoxic effect in normal cells and inhibited the growth of HT-29 cells depending on concentration and incubation time. PLE-treated HT-29 cells showed the typical morphological characteristics of apoptosis, such as apoptotic body formation and DNA fragmentation. PLE also induced mitochondrial membrane potential depolarization and resulted in increased mitochondrial membrane permeability, compared with untreated cells. PLE decreased Bcl-2 protein and increased Bax protein expression, activating caspase-3 and poly (ADP-ribose) polymerase (PARP) expression via the caspase pathway. PLE also increased the phosphorylation of c-Jun N-terminal kinase (JNK), p38, and extracellular signal-regulated kinase (ERK), and it reduced cell viability in treatment cells with specific inhibitors such as PD98059 (a specific inhibitor of ERK), SP600125 (a specific inbibitor of JNK), and SB 203580 (a specific inbibitor of p38 MAPK). via the the mitogen-activated protein kinases (MAPKs) pathway. These results suggest that PLE inhibits the proliferation of HT-29 cells by affecting the caspase and MAPK pathways involved in the induction of apoptosis. Thus, we suggest that P. littoralis extract might be potential candidate agents for the treatment of human colorectal cancer.