Hookworms digest hemoglobin from erythrocytes via a proteolytic cascade that begins with the aspartic protease, APR-1. Ac-APR-1 from the dog hookworm, Ancylostoma caninum, protects dogs against hookworm infection via antibodies that neutralize enzymatic activity and interrupt blood-feeding. Toward developing a human hookworm vaccine, we expressed both wild-type (Na-APR-1(wt)) and mutant (Na-APR-1(mut)-mutagenesis of the catalytic aspartic acids) forms of Na-APR-1 from the human hookworm, Necator americanus. Refolded Na-APR-1(wt) was catalytically active, and Na-APR-1(mut) was catalytically inactive but still bound substrates. Vaccination of canines with Na-APR-1(mut) and heterologous challenge with A. caninum resulted in significantly reduced parasite egg burdens (P=0.034) and weight loss (P=0.022). Vaccinated dogs also had less gut pathology, fewer adult worms, and reduced blood loss compared to controls but these did not reach statistical significance. Vaccination with Na-APR-1(mut) induced antibodies that bound the native enzyme in the parasite gut and neutralized enzymatic activity of Na-APR-1(wt) and APR-1 orthologues from three other hookworm species that infect humans. IgG1 against Na-APR-1(mut) was the most prominently detected antibody in sera from people resident in high-transmission areas for N. americanus, indicating that natural boosting may occur in exposed humans. Na-APR-1(mut) is now a lead antigen for the development of an antihematophagy vaccine for human hookworm disease.