AeSCP-2, a sterol carrier protein, is involved in sterol trafficking in mosquitoes. The activity of the AeSCP-2 gene is important for mosquito development. An earlier study demonstrated that the transcription of this gene was upregulated by 20-hydroxyecdysone (20E) in cultured gut tissues. To investigate 20E-regulated transcription of the AeSCP-2 gene we truncated the upstream flanking region of AeSCP-2 gene and linked it to a reporter gene. The mosquito Aag-2 cell line was transfected with these promoter/reporter constructs and treated with 20E at various concentrations. Expression vectors of different transcription factors such as HR3 and βFTZ-F1 were also co-transfected with the AeSCP-2 promoter/reporter constructs. The observed results demonstrated that varied combinations of transcription factors produce different promoter activities of the AeSCP-2 gene. This observation leads us to the conclusion that the partnership of transcription factors is crucial in regulating the transcriptional activity of the AeSCP-2 gene.