Aquaporin adipose (AQPap) is a putative glycerol channel in adipocytes (Kishida, K., Kuriyama, H., Funahashi, T., Shimomura, I., Kihara, S., Ouchi, N., Nishida, M., Nishizawa, H., Matsuda, M., Takahashi, M., Hotta, K., Nakamura, T., Yamashita, S., Tochino, Y., and Matsuzawa, Y. (2000) J. Biol. Chem. 275, 20896-20902). In the current study, we examined the genomic structure of the mouse AQPap gene and its regulation by insulin. The mouse AQPap gene spanned 12 kilobase pairs in chromosome 4 and consisted of 8 exons and 7 introns. The first two exons, designated exon 1 and exon 1', are alternatively spliced to common exon 2, and thus the AQPap gene possessed two potential promoters. The exon 1-derived transcript is dominant in both adipose tissues and adipocytes on the basis of RNase protection assay and promoter analysis. The mRNA increased after fasting and decreased with refeeding. Insulin deficiency generated by streptozotocin enhanced the mRNA in adipose tissue. Insulin down-regulated AQPap mRNA in 3T3-L1 adipocytes. The AQPap promoter contained heptanucleotide sequences, TGTTTTT at -443/-437, similar to the insulin-response element identified previously in the promoters of insulin-repressed genes. Deletion and single base pair substitution analysis of the promoter revealed that these sequences were required for insulin-mediated repression of AQPap gene transcription. The phosphatidylinositol 3-kinase pathway was involved in this inhibition. We conclude that insulin represses the transcription of AQPap gene via insulin response element in its promoter. Sustained up-regulation of AQPap mRNA in adipose tissue in the insulin-resistant condition may disturb glucose homeostasis by increasing plasma glycerol.