The malaria parasite Plasmodium falciparum degrades host cell hemoglobin within an acidic food vacuole. The metalloprotease falcilysin has previously been identified as an important component of this catabolic process. Using random peptide substrate analysis, we confirm that recombinant falcilysin is highly active at acidic pH, consistent with its role in hemoglobin degradation. Unexpectedly, the enzyme is also robustly active at neutral pH, but with a substantially different substrate specificity. Imaging studies confirm the location of falcilysin in the food vacuole and reveal association with vesicular structures elsewhere within the parasite. These data suggest that falcilysin may have an expanded role beyond globin catabolism and may function as two different proteases in its two locations.