The repat gene family encodes midgut proteins overexpressed in response to pathogen infection in the lepidopteran Spodoptera exigua. Up-regulation of repat genes has been observed after challenging the larvae with both Bacillus thuringiensis toxins and after infection with the baculovirus Autographa californica multiple nucleopolyhedrovirus. In our study, PCR amplification of the genomic region and genome walking were used to obtain the genomic structure and the sequence of the 5′-upstream region of repat1 and repat2, two of the most phylogenetically distant members of the repat family. A similar gene structure between repat1 and repat2 has been found, with conserved exon-intron positions and junction sequences, suggesting a common origin for these genes. Recombinant baculoviruses carrying the firefly luciferase gene under the control of different 5′-upstream regions of the repat1 gene were constructed to elucidate the influence of these regions in gene expression. Infection of Helicoverpa zea gut-derived cells with the recombinant baculoviruses revealed the upstream regions of the repat1 gene which are involved in gene transcription and demonstrated the role of an intron located in the 5′-untranslated region in the enhancement of gene expression.