Type I signal peptidases are important membrane-bound serine proteases responsible for the cleavage of the signal peptide of the proteins. These enzymes are unique serine proteases that carry out catalysis using a serine/lysine catalytic dyad. In the present study, we report the isolation of type I signal peptidase from the malaria parasites Plasmodium falciparum, Plasmodium knowlesi, and Plasmodium yoelii and some characterization of type I signal peptidase of Plasmodium falciparum. We show that these enzymes are homologous to signal peptidases from various sources and also contain the conserved boxes present in other type I signal peptidases. The type I signal peptidase from P falciparum is an intron-less and a single-copy gene. The results also show that the enzyme from Plasmodium falciparum is subject to self-cleavage and it has been demonstrated to possess type I signal peptidase activity in E coli preprotein processing in vivo by complementation assay. This study will be helpful in understanding one of the important metabolic pathways “the secretory pathway” in the parasite and should make an important contribution in understanding the complex process of protein targeting in the parasite.