Choline transporter-like (CTL) proteins of the CTL1 family are novel transmembrane proteins implicated in choline transport for phospholipid synthesis. In this study, we characterized the 5′-flanking region of the human (h)CTL1 gene and examined some of the possible mechanisms of its regulation, including promoter activity, splicing, and expression. The transcription start site of the hCTL1 gene was mapped by 5′-rapid amplification of cDNA ends (RACE), and the presence of two splice variants, hCTL1a and hCTL1b, was investigated using isoform-specific PCR and 3′-RACE. The hCTL1 promoter region of ∼900 bp was isolated from MCF-7 human breast cancer cells. The promoter was TATA-less and driven by a long stretch of GC-rich sequence in accordance with widespread expression of hCTL1 at both mRNA and protein levels. Deletion analyses demonstrated that a very strong promoter is contained within 500 bp of the transcription start site, and more upstream regions did not increase its activity. The core promoter that conferred the minimal transcription is within the −188/+27-bp region, and its activity varied in human breast cancer and mouse skeletal muscle cells. Multiple motifs within the promoter regulatory region bound nuclear factors from both cultured cells and normal human skeletal muscle. The motifs within the three regions [S1 (−92/−61 bp), S2 (−174/−145 bp), and S3 (−289/−260 bp)] contained overlapping binding sites for hematopoietic transcription factors and ubiquitous transcription factors, in line with the expected gene function. Genomic analyses demonstrated a high conservation of hCTL1 and mouse CTL1 proximal promoters. Accordingly, mRNA profiles demonstrated that human splice variants were expressed ubiquitously, as demonstrated for the mouse transcripts; however, they differed from the profiles of rat CTL1 transcripts, which were more restricted to neurons and intestinal tissues. The shorter hCTL1b variant contained the cytosolic COOH-terminal motif L651KKR654for endoplasmic reticulum retrieval/retention. This retention signal was conserved in hCTL1b and rat and mouse CTL1b and is typical for transmembrane proteins of type 1 topology.