Gamma-interferon-inducible lysosomal thiol reductase (GILT) has been demonstrated to be involved in the immune response to bacterial challenge in various organisms. However, little is known about GILT function in innate immunity. Drosophila has been commonly used as a model for the study of the innate immune response of invertebrates. Here, we identify the CG9796, CG10157, and CG13822 genes of fruit fly Drosophila melanogaster as GILT homologues. All deduced Drosophila GILT coding sequences contained the major characteristic features of the GILT protein family: the GILT signature CQHGX2ECX2NX4C sequence and the active site CXXC or CXXS motif. The mRNA transcript levels of the Drosophila GILT genes were up-regulated after Gram-negative bacteria Escherichia coli DH5α infection. Moreover, a bacterial load assay showed that over-expression of Drosophila GILT in fat body or hemocytes led to a low bacterial colony number whereas knock-down of Drosophila GILT in fat body or hemocytes led to a high bacterial colony number when compared to a wild-type control. These results indicate that the Drosophila GILTs are very likely to play a role in the innate immune response upon bacterial challenge of Drosophila host defense. This study may provide the basis for further study on GILT function in innate immunity.