Hookworms infect more than 700 million people worldwide and cause more morbidity than most other human parasitic infections. Nippostrongylus brasiliensis (the rat hookworm) has been used as an experimental model for human hookworm because of their similar life cycles and ease of maintenance in laboratory rodents. Adult N. brasiliensis, like the human hookworm, live in the intestine of the host and release excretory/secretory products (ESP), which represent the major host-parasite interface. We performed a comparative proteomic analysis of infective larval (L3) and adult worm stages of N. brasiliensis to gain insights into the molecular bases of host-parasite relationships and determine whether N. brasiliensis could indeed serve as an appropriate model for studying human hookworm infections. Proteomic data were matched to a transcriptomic database assembled from 245,874,892 Illumina reads from different developmental stages (eggs, L3, L4 and adult) of N. brasiliensis yielding ~18,426 unigenes with 39,063 possible isoform transcripts. From this analysis, 313 proteins were identified from ESPs by LC-MS/MS - 52 in the L3 and 261 in the adult worm. Most of the proteins identified in the study were stage-specific (only 13 proteins were shared by both stages); in particular two families of proteins - astacin metalloproteases and CAP-domain containing SCP/TAPS - were highly represented in both L3 and adult ESP. These protein families are present in most nematode groups, and where studied, appear to play roles in larval migration and evasion of the host's immune response. Phylogenetic analyses of defined protein families and global gene similarity analyses showed that N. brasiliensis is more closely related to human hookworm than are other model nematodes including the murine gastrointestinal parasite Heligmosomoides polygyrus. These findings validate the use of N. brasiliensis as a suitable parasite for the study of human hookworm infections in a tractable animal model.