Hookworms are blood-feeding intestinal parasites of mammalian hosts and are one of the major human ailments affecting approximately 600 million people worldwide. These parasites form an intimate association with the host and are able to avoid a vigorous immune responses in many ways including skewing of the response phenotype to promote parasite survival and longevity. The primary interface between the parasite and the host is the excretory/secretory component; a complex mixture of proteins, carbohydrates and lipids secreted from the surface or oral openings of the parasite. The composition of this complex mixture is for the most part unknown but is likely to contain proteins important for the parasitic lifestyle and hence suitable as drug or vaccine targets. Using a strategy combining the traditional technology of 1D SDS-PAGE and the newer fractionation technology of OFFGEL electrophoresis we identified 105 proteins from the blood-feeding stage of Ancylostoma caninum. Highly represented among the identified proteins where lectins, including three C-type lectins and three ß–galactoside-specific S-type galectins, as well as a number of proteases, including new proteases belonging to the three major classes found in nematodes – aspartic, cysteine and metallo-proteases. Interestingly, 28% of the identified proteins were homologous to Activation-associated secreted proteins, a family of cysteine-rich secreted proteins belonging to the SCP/TAPS super-family. Thirty-four of these proteins were identified suggesting an important role in host/parasite interactions. Other protein families identified included hyaluronidases, lysozyme-like proteins and transthyretin-like proteins. This work identifies a suite of proteins important for the parasitic lifestyle and provides new insight into the biology of hookworm infection.