Superoxide dismutases (SOD) are a family of antioxidant enzymes that function by removing superoxide anions from the cellular environment. Here, we show that the African trypanosome, Trypanosoma brucei, expresses four SOD isoforms, three of which we have validated biochemically as iron dependent, a feature normally associated with prokaryotic SODs. Localisation studies reveal that two of the enzymes are found predominantly in a parasite-specific organelle, the glycosome (TbSODB1 and TbSODB2), while the other two are targeted to the mitochondrion (TbSODA and TbSODC). Functional analysis of the SOD repertoire in bloodstream form parasites was performed using an inducible RNA interference (RNAi) approach. Down-regulation of the glycosomal SOD transcripts corresponded with a significant reduction in the corresponding proteins and a dramatic level of cell death within the population. The importance of one of the mitochondrial enzymes (TbSODA) only became apparent when parasites were exposed to the superoxide-generating agent paraquat following induction of RNAi. These experiments therefore identify essential components of the superoxide metabolising arm of the T. brucei oxidative defence system and validate these enzymes as parasite-specific targets for drug design.