<p>Non-alcoholic fatty liver disease (NAFLD) is a chronic condition with excess accumulation of fat in the liver, affecting approximately 25% of the global population. It involves complex crosstalk between multiple organs, yet current in vitro models inadequately capture this multi-organ interaction. We developed a modular multi-organ chip system consisting of the gut, liver, and adipose tissue, the organs involved in lipid absorption, metabolism, and storage. This platform enables the co-culture of separately differentiated intestinal (Caco-2), hepatic (HepG2), and adipose (3T3-L1) cells under gravity-driven flow conditions to investigate lipid metabolism and inter-organ communication. The cells maintained high cell viability and proper adipocyte differentiation comparable to conventional static culture models. We assessed lipid accumulation following fatty acid treatment in the intestinal compartment under normal conditions and barrier-compromised conditions induced by lipopolysaccharide (LPS) and dextran sulfate sodium (DSS). Disrupted intestinal barrier integrity significantly enhanced lipid translocation and accumulation in both hepatic and adipose tissues compared to intact epithelium, with adipose cells exhibiting substantially greater lipid accumulation than hepatic cells, reflecting their specialized role in lipid storage. Nitric oxide (NO) measurements across all compartments consistently revealed particularly high levels in the intestinal compartment, with tissue-specific responses to inflammatory stimulation. Our integrated gut–liver–adipose tissue model successfully recapitulates key aspects of inter-organ metabolic crosstalk and demonstrates that intestinal barrier integrity critically influences tissue-specific lipid deposition. This approach offers unique advantages over conventional single-organ models for investigating the multifactorial mechanisms underlying metabolic disorders, such as NAFLD, and for developing therapeutic strategies targeting multiple organ systems.</p>