Abstract: Triclosan (TCS), an antimicrobial additive, has been widely used in many daily consumer products. Although it is banned from using in soap products in 2016 by the USA Food and Drug Administration, it still can be used in many other products such as cosmetics, toys, mouthwash and toothpaste. Because of its ubiquitous characteristics, there is a growing concern over the impact of TCS on human health. Previous studies have reported that exposure to TCS could promote the development of hepatocellular carcinoma. However, little is known about the related mechanisms at the metabolic levels. In cancer research, multicellular tumor spheroids (MCTSs) are sphereshaped cellular colonies, and which are considered to be the most appropriate in vitro cell model to test toxicities of chemicals because of their ability to mimic the main characteristics of solid tumors. To the best of our knowledge, no studies have tested the effect of TCS on the development of liver MCTS. Hence, in this work, liver MCTSs were exposed to TCS, and mass spectrometry imaging (MSI) was used to explore the variations of abundance and distribution of lipids in MCTS between exposure and control groups. The results revealed that treatment of 6 μmol/L TCS could significantly promote the growth of liver MCTS and induce the lipid disorders in MCTS. Segmentation analysis of MCTS showed that there were three microregions including the outer proliferative area, the middle quiescent area and the inner necrotic area in MCTS. Further MSI data revealed that the abundance of 27 lipids including 19 glycerophospholipids (GPs), 3 glycerolipids (GLs) and 5 sphingolipids (SPs) significantly changed in MCTS between treated and untreated groups. In GPs, a total of 16 upregulated lipids located in the outer proliferative area of MCTS. In GLs, two upregulated lipids located in the outer proliferative area, while one downregulated lipid distributed in the inner area. In SPs, all five lipids with decreased levels located in the inner area. All these data suggested that the enhanced growth of liver MCTS induced by treatment of TCS may through promoting the growth of cells in the outer region of MCTS and inhibiting the death of cells in the inner region of MCTS. This result may offer a better understanding for the mechanisms of the effects of environmental pollutants on cancer progression.