Malaria is the world’s most prevalent parasitic disease, with over 200 million cases and 627,000 resultant deaths annually. Alarmingly, the spread of drug-resistant parasites has rendered most antimalarials ineffective, and has made the development of novel therapeutic strategies a global health priority. Malarial parasites have a complicated lifecycle involving an asymptomatic ‘liver stage’ and a symptomatic ‘blood stage’. During the blood stage, the parasites utilise a proteolytic cascade to digest host haemoglobin, which produces free amino acids absolutely necessary for parasite growth and reproduction. The enzymes required for haemoglobin digestion are therefore attractive therapeutic targets. The final step of the cascade is catalyzed by several metalloaminopeptidases, including prolyl aminopeptidase P (APP). We have determined the crystal structure of APP from P. falciparum (PfAPP), and present the first examination of the 3D structure of this important malarial enzyme. Further, we developed a chemical genetics platform to examine the substrate fingerprint of PfAPP, which provides insight into potential mechanisms of inhibition that could be used to develop novel antimalarial therapeutics.