The Drosophila gene, pixie, is an essential gene required for normal growth and translation. Pixie is the fly ortholog of human RLI, which was first identified as an RNase L inhibitor, and yeast Rli1p, which has recently been shown to play a role in translation initiation and ribosome biogenesis. These proteins are all soluble ATP-binding cassette proteins with two N-terminal iron-sulfur clusters. Here we demonstrate that Pixie can be isolated from cells in complex with eukaryotic translation initiation factor 3 and ribosomal proteins of the small subunit. In addition, our analysis of polysome profiles reveals that double-stranded RNA interference-mediated depletion of Pixie results in an increase in empty 80 S ribosomes and a corresponding decrease in polysomes. Thus Pixie is required for normal levels of translation initiation. We also find that Pixie associates with the 40 S subunit on sucrose density gradients in an ATP-dependent manner. Our observations are consistent with Pixie playing a catalytic role in the assembly of complexes required for translation initiation. Thus, the function of this soluble ATP-binding cassette domain protein family in translation initiation has been conserved from yeast through to higher eukaryotes.