Background: Visceral adipose tissue (VAT) and subcutaneous adipose tissue (SCAT) have different structures and metabolic functions, and play different roles in the regulation of mammal systemic endocrine. However, little is known about morphology and physiological and metabolic functions between VAT and SCAT in fish. Methods: We compared the morphological, physiological and biochemical characteristics of VAT and SCAT in Nile Tilapia and measured their functions in energy intake flux, lipolytic ability, and gene expression patterns. Results: SCAT contained more large adipocytes and non-adipocytes than VAT in Nile tilapia. VAT had a higher lipid and was the primary site for lipid deposition. Conversely, SCAT had higher hormone-induced lipolytic activity. Furthermore, SCAT had a higher percentage of monounsaturated and lower polyunsaturated fatty acids than VAT. SCAT had higher mitochondrial DNA, gene expression for fatty acid β-oxidation, adipogenesis and brown adipose tissue characteristics, but it also had a lower gene expression for inflammation and adipocyte differentiation than VAT. Conclusions: SCAT and VAT have different morphological structures, as well as physiological and metabolic functions in fish. VAT is the preferable lipid deposition tissue, whereas SCAT exhibits higher lipid catabolic activity than VAT. General Significance: The physiological functions of SCAT in fish are commonly overlooked. The present study indicates SCAT has specific metabolic characteristics that differ VAT. The differences between VAT and SCAT should be considered in future metabolism studies using fish as models, either in biomedical or aquaculture studies.