Galanin and its receptors have been shown to be expressed in undifferentiated mouse embryonic stem (ES) cells through transcriptome and proteomic analyses. Although transcriptional regulation of galanin has been extensively studied, the regulatory proteins that mediate galanin expression in mouse ES cells have not yet been determined. Through sequence alignments, we have found a high degree of similarity between mouse and human galanin upstream sequences at −146 bp/+69 bp (proximal region) and −2408 bp/−2186 bp (distal region). These regions could be recognized by ES cell nuclear proteins, and EMSA analysis suggests a specific functionality. Analysis of the proximal region (PR) using EMSA and ChIP assays showed that the CREB protein interacts with the galanin promoter both in vitro and in vivo. Additional EMSA analysis revealed that an SP1 consensus site mediated protein–DNA complex formation. Reporter assays showed that CREB is an activator of galanin expression and works cooperatively with SP1. Furthermore, analysis of the distal region (DR) using EMSA assays demonstrated that both HOX-F and PAX 4/6 consensus sites mediated protein–DNA complex formation, and both sites inhibited luciferase activity in reporter assays. These data together suggest that CRE and SP1 act as activators at the basal promoter, while HOX-F and PAX 4/6 act as silencers of transcription. The interplay of these transcription factors (TF) may drive regulated galanin expression in mouse ES cells.