Insulin induction of glucokinase (GCK) transcription in the liver is essential for maintaining glucose homeostasis. To study the molecularmechanism underlying the regulation of hepatic GCK expression in the carnivorous fish gilthead sea bream (Sparus aurata), we analysedthe role of sterol regulatory element binding protein-1a (SREBP-1a) and specificity protein (Sp) 1 in insulin-dependent GCKtranscription. Transient transfection experiments performed in HepG2 cells and electrophoretic mobility shift assays allowed us to identifya cis-element in the proximal region of GCK promoter implicated in transactivation by SREBP-1a. Consistently, mutations in theSRE binding site completely abolished the enhancing effect of SREBP-1a. These results and previous findings suggest that SREBP-1aplays a role in the transcriptional regulation of key enzymes in glycolysis–gluconeogenesis. Since SREBP-1a and Sp1 may mediate insulinaction on S. aurata GCK transcription, we analysed the effect of insulin on HepG2 cells transfected with GCK promoter reporter constructscarrying intact or mutated SRE or Sp boxes. Insulin transactivated GCK irrespective of the presence of an intact or mutated SREbox. However, insulin failed to induce GCK transcription when using reporter constructs that had either a mutated Sp site or no Sp site.Our findings indicate that Sp1, rather than SREBP-1a, mediates the insulin-dependent induction of S. aurata GCK.