Systemic adipose tissue is involved in the pathophysiology of obesity-associated liver diseases. However, a method has not been established for analyzing the direct interaction between adipose tissue and hepatocytes. We describe a useful three-dimensional model comprising a collagen gel coculture system in which HepG2 hepatocytes are cultured on a gel layer with visceral adipose tissue fragments (VAT) or subcutaneous tissue samples (SAT). Male adipose tissues were obtained from 5-week-old Wistar rats and human autopsy cases. Cellular behavior was analyzed by electron microscopy, immunohistochemistry, Western blot, real-time reverse transcription plus the polymerase chain reaction and enzyme-linked immunosorbent assay. VAT significantly promoted lipid accumulation and apoptosis in HepG2 cells and suppressed their growth and differentiation compared with SAT. VAT produced higher concentrations of fatty acids (palmitate, oleate, linoleate) than SAT. HepG2 cells significantly decreased the production of these fatty acids in VAT. Only HepG2 cells treated with 250 μM palmitate replicated VAT-induced apoptosis. Neither VAT nor SAT affected lipotoxicity-associated signals of nuclear factor kappa B, c-Jun N-terminal kinase and inositol requiring enzyme-1α in HepG2 cells. HepG2 cells never affected adiponectin, leptin, or resistin production in VAT and SAT. The data indicate that our model actively creates adipose tissue and HepG2 hepatocyte interactions, suggesting that (1) VAT plays more critical roles in hepatocyte lipotoxicity than SAT; (2) palmitate but not adipokines, is partly involved in the mechanisms of VAT-induced lipotoxicity; (3) HepG2 cells might inhibit fatty acid production in VAT to protect themselves against lipotoxicity. Our model should serve in studies of interactions between adipose tissue and hepatocytes and of the mechanisms in obesity-related lipotoxicity and liver diseases.