Orexins, acting via their receptors, are involved in the control of feeding, sleep-wakefulness, arousal, neuroendocrine homeostasis and autonomic regulation. However, the 5′ structure and regulation of human orexin type 2 receptor (OX2R) gene remains is not known. We present original findings regarding the 5′ structural organization of the human OX2R gene and identify four OX2R mRNA transcripts that differ in their 5′-untranslated region (UTR). The four transcripts revealed that the three alternative exons arise from alternative splicing. These exon 1 variants, arising from a single OX2R gene, were distributed over a region of 29504 bp and designated as exons 1A, 1B and 1C on the basis of their 5′ to 3′ order. In transfection studies, different transcripts exerted cell-specific effects on mRNA, but consistently reduced protein expression. Tissue-specific expression of these transcripts in human tissues has been demonstrated by RT-PCR. We show those 5′ -flanking regions to exon 1A and exon 2, but not exon 1C, drive alternative promoter activity in HEK-293 and SH-5YSY cells. Using progressive deletion analysis, a proximal promoter region between −456 and −123 (relative to the translation start site) was shown to exhibit the higher activities in HEK-293, SH-5YSY and NT2 cells. One CRE, GATA-2 and Oct-1 motif was identified within this region, which was responsible for the stimulation both by Dibutyryil-cAMP (db cAMP) and phorbol-12-myristate-13-acetate (PMA). Mutational studies demonstrated that these motifs functioned co-operatively to stimulate hOX2R gene transcription. Using the chromatin immunoprecipitation assay, we demonstrated that three motifs bind to the region of hOX2R proximal promoter. These novel data suggest that usage of alternate promoters, 5′-UTR and alternative splicing may contribute regulatory mechanisms for tissue-specific expression of the hOX2R gene.