Proteins containing the 21st amino acid selenocysteine (Sec) are present in the three domains of life. However, within lower eukaryotes, particularly parasitic protists, the dependence on the trace element selenium is variable as many organisms lost the ability to utilize Sec. Herein, we analyzed the genomes of Trypanosoma and Leishmania for the presence of genes coding for Sec-containing proteins. The selenoproteomes of these flagellated protozoa have three selenoproteins, including distant homologs of mammalian SelK and SelT, and a novel multidomain selenoprotein designated SelTryp. In SelK and SelTryp, Sec is near the C-terminus, and in all three selenoproteins, it is within predicted redox motifs. SelTryp has neither Sec- nor cysteine-containing homologs in the human host and appears to be a Kinetoplastida-specific protein. The use of selenium for protein synthesis was verified by metabolically labeling Trypanosoma cells with ⁷⁵Se. In addition, genes coding for components of the Sec insertion machinery were identified in the Kinetoplastida genomes. Finally, we found that Trypanosoma brucei brucei cells were highly sensitive to auranofin, a compound that specifically targets selenoproteins. Overall, these data establish that Trypanosoma, Leishmania and likely other Kinetoplastida utilize and depend on the trace element selenium, and this dependence is due to occurrence of selenium in at least three selenoproteins.