Alanine aminotransferase (ALT) plays an important role in amino acid metabolism and gluconeogenesis. The preference of carnivorous fish for protein amino acids instead of carbohydrates as a source of energy lead us to study the transcriptional regulation of the mitochondrial ALT (mALT) gene and to characterize the enzyme kinetics and modulation of mALT expression in the kidney of gilthead sea bream (Sparus aurata) under different nutritional and hormonal conditions. 5′-Deletion analysis of mALT promoter in transiently transfected HEK293 cells, site-directed mutagenesis and electrophoretic mobility shift assays allowed us to identify HNF4α as a new factor involved in the transcriptional regulation of mALT expression. Quantitative RT-PCR assays showed that starvation and the administration of streptozotocin (STZ) decreased HNF4α levels in the kidney of S. aurata, leading to the downregulation of mALT transcription. Analysis of the tissue distribution showed that kidney, liver, and intestine were the tissues with higher mALT and HNF4α expression. Kinetic analysis indicates that mALT enzyme is more efficient in catalyzing the conversion of L-alanine to pyruvate than the reverse reaction. From these results, we conclude that HNF4α transactivates the mALT promoter and that the low levels of mALT expression found in the kidney of starved and STZ-treated fish result from a decreased expression of HNF4α. Our findings suggest that the mALT isoenzyme plays a major role in oxidazing dietary amino acids, and points to ALT as a target for a biotechnological action to spare protein and optimize the use of dietary nutrients for fish culture.