An unfractionated fucoidan was extracted from the brown alga Ascophyllum nodosum. Extraction of fucoidan from seaweed was carried out using an innovative low-chemical process. A combinational approach involving compositional analysis, HPAEC, IR analysis, GPC, and NMR was employed to elucidate the composition and structure of an unfractionated fucoidan from A. nodosum. This fucoidan is composed mainly of fucose (52.1%), and also galactose (6.1%), glucose (21.3%), and xylose (16.5%). Sulfate content was determined to be 19%. GPC data indicated a polydisperse fucoidan containing two main size fractions (47 and 420 kDa). NMR analyses revealed a fucoidan displaying broad, complex signals as expected for such a high molecular weight and heterogeneous polymer with resonances consistent with a fucoidan isolated previously from A. nodosum. The effects of fucoidan on the apoptosis of human colon carcinoma cells and fucoidan-mediated signaling pathways were also investigated. Fucoidan decreased cell viability and induced apoptosis of HCT116 colon carcinoma cells. Fucoidan treatment of HCT116 cells induced activation of caspases-9 and -3 and the cleavage of PARP, led to apoptotic morphological changes, and altered mitochondrial membrane permeability. These results detail the structure and biological activity of an unfractionated fucoidan from A. nodosum.