Ultraviolet A (UVA) radiation, the major component of solar ultraviolet (UV) radiation reaching the earth's surface, leads to negative effects in insects, such as oxidative stress, photoreceptor damage, and cell death. To better understand the molecular mechanisms of insect response to UVA radiation, suppression subtractive hybridization (SSH) and real-time quantitative polymerase chain reaction approaches were combined to reveal differential transcript expression in Drosophila melanogaster Meigen, 1830 (Diptera: Drosophilidae). In this study, two subtractive cDNA libraries were constructed and sequenced, obtaining 131 high-quality unique expressed sequence tags (ESTs) that were up- or downregulated in D. melanogaster exposed to UVA radiation for 0.5 h. Of the 131 ESTs, 102 unique ESTs were differentially expressed and classified into 10 functional categories. The results showed that UVA radiation induces expression of genes related to stress and defense response and metabolism. Potential transcription factor binding motifs upstream of these genes are associated with multiple signaling pathways that may help the insect cope with the stress of UVA radiation. To our knowledge, this is the first analysis of insect response to UVA radiation at the transcriptional level. Our results reveal that UVA radiation influences the expression profiles of stress-responsive genes and provide further insights into the mechanisms of adaptive response to UVA radiation stress.