Three-dimensional (3D) cell cultures offer more physiologically relevant models than conventional cell monolayers (2D) for mimicking in vivo tissue architecture. Despite their advantages, the use of 3D fish cell models in toxicological studies remains limited. This study aims to characterize the lipidomic and gene expression profiles of zebrafish liver (ZFL) spheroids and evaluate their metabolic and functional differences compared to those of conventional monolayer cultures. Seven-day-old spheroids (400 μm diameter) exhibited increased levels of neutral lipidscholesterol esters (CEs) and diacylglycerols (DGs)alongside reduced expression of genes involved in fatty acid synthesis (fasn, acc, and scebf1) compared to cell monolayers. This suggests a metabolic shift from lipid synthesis to lipid storage. Additionally, spheroids showed a reorganization of membrane lipids, with an increased ratio of phosphatidylcholines (PCs) to phosphatidylethanolamines (PEs) and elevated levels of phospholipids containing long-chain polyunsaturated fatty acids such as docosahexaenoic acid (DHA, 22:6). Upon exposure to β-naphthoflavone (BNF), the spheroids showed a markedly higher expression of cyp1a (650-fold) compared to monolayers (57-fold increase), indicating increased metabolic competence. Overall, ZFL spheroids represent a more mature and functionally relevant hepatocyte model than cell monolayers, particularly in terms of lipid composition and metabolic function. The study highlights the importance of lipidomic studies in characterizing 3D models and provides valuable insights into molecular changes associated with spheroid maturation.