Phosphatidylserine (PS) is an essential constituent of biological membranes and plays critical roles in apoptosis and cell signaling. Because no information was available on transcriptional mechanisms that regulate PS biosynthesis in mammalian cells, we investigated the regulation of expression of the mouse PS synthase-1 (Pss1) gene. The Pss1 core promoter was characterized in vitro and in vivo through gel shift and chromatin immunoprecipitation assays. Transcription factor-binding sites, such as a GC-box cluster that binds Sp1/Sp3/Sp4 and N-Myc, and a degenerate E-box motif that interacts with Tal1 and E47, were identified. Pss1 transactivation was higher in brain of neonatal mice than in other tissues, consistent with brain being a major site of expression of Pss1 mRNA and PSS1 activity. Enzymatic assays revealed that PSS1 activity is enriched in primary cortical astrocytes compared with primary cortical neurons. Site-directed mutagenesis of binding sites within the Pss1 promoter demonstrated that Sp and N-Myc synergistically activate Pss1 expression in astrocytes. Chromatin immunoprecipitation indicated that Sp1, Sp3, and Sp4 interact with a common DNA binding site on the promoter. Reduction in levels of Sp1, Sp3, or N-Myc proteins by RNA interference decreased promoter activity. In addition, disruption of Sp/DNA binding with mithramycin significantly reduced Pss1 expression and PSS1 enzymatic activity, underscoring the essential contribution of Sp factors in regulating PSS1 activity. These studies provide the first analysis of mechanisms that regulate expression of a mammalian Pss gene in brain.