The pathogenesis of malarial anemia is incompletely understood. Hepcidin, a recently discovered peptide hormone, is a major regulator of iron metabolism and is thought to play a central role in the anemia of chronic inflammation. The specific aim of the study was to characterize the association between urinary hepcidin, hemoglobin, and parasitemia in 199 patients presenting for evaluation of Plasmodium falciparum malaria in Ghana. Urinary hepcidin was semi-quantitatively assessed using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry. Urinary hepcidin (intensity/mmol creatinine) was associated with log parasitemia in 86 children (beta = 0.086, standard error [SE] = 0.035, P < 0.017), 31 pregnant women (beta = 0.218, SE = 0.085, P < 0.016), and 82 adults (beta = 0.184, SE =0.043, P < 0.0001). Urinary hepcidin was not significantly associated with hemoglobin or anemia. Urinary hepcidin is more strongly associated with parasitemia than hemoglobin or anemia among patients with acute P. falciparum malaria in Ghana.