Liver models that closely mimic the in vivo microenvironment are central for understanding liver functions, capabilities, and intercellular communication processes. Whereas hepatocyte monolayers de-differentiate after only a few days in culture, constructs consisting of hepatocytes and non-parenchymal cells (NPC) separated by a polyelectrolyte multilayer (PEM) provide maintenance of hepatic phenotypes. To better understand the mechanisms and processes that underlie organotypic liver model function, hepatocyte protein abundances in the presence and absence of liver sinusoidal endothelial cells (LSECs) were compared to hepatocyte monolayers. The presence of the PEM led to increases in proteins associated with hepatocyte lipid metabolism, with mitochondrial-based β-oxidation and peroxisomal proteins found in higher abundance. It was also demonstrated that the presence of LSECs modulates levels of carboxylesterases and other phase I and phase II enzymes. These results are discussed in relation to intercellular signaling and hepatocyte function.