Glycerol-3-phosphate acyltransferase 1 (GPAT1) is a rate limiting enzyme in de novo glycerophospholipid synthesis. The murine GPAT1 promoter sequence (the "classical" sequence) was reported previously. However, the organization of this DNA sequence does not fully match the mouse genome sequences on NCBI/GenBank. Here we have identified net cis-acting promoter sequences for the mouse GPAT1 gene: promoter 1a which includes part of the classical sequence and the downstream promoter 1b. Promoter 1a facilitates transcription of two alternative GPAT1 transcript variants, GPAT1-V1 and V2, while promoter 1b produces a third transcript variant, GPAT1-V3. Upstream stimulating factor-1 (USF-1) controlled both promoters whereas sterol regulatory element-binding protein-1 (SREBP-1) exclusively regulated promoter 1a activity in vitro. Feeding increased GPAT1-V1 and V2, but not V3 mRNA levels in mouse liver. The obese condition of db/db mice did not alter the hepatic expression levels of any of the three GPAT1 variants. Feeding enhanced hepatic mRNA levels, intranuclear protein levels and promoter 1a-binding levels of SREBP-1, but not of USF-1. Thus, promoter 1a was exclusively activated by routine feeding in vivo. Our results indicate differential roles of the two promoters in the regulation of hepatic GPAT1 gene expression in mice.