Liver homeostasis requires the presence of both parenchymal and non-parenchymal cells (NPCs). However, systems biology studies of the liver have primarily focused on hepatocytes. Using an organotypic three-dimensional (3D) hepatic culture, we report the first transcriptomic study of liver sinusoidal endothelial cells (LSECs) and Kupffer cells (KCs) cultured with hepatocytes. Through computational pathway and interaction network analyses, we demonstrate that hepatocytes, LSECs and KCs have distinct expression profiles and functional characteristics. Our results show that LSECs in the presence of KCs exhibit decreased expression of focal adhesion kinase (FAK) signaling, a pathway linked to LSEC dedifferentiation. We report the novel result that peroxisome proliferator-activated receptor alpha (PPAR alpha) is transcribed in LSECs. The expression of downstream processes corroborates active PPAR alpha signaling in LSECs. We uncover transcriptional evidence in LSECs for a feedback mechanism between PPAR alpha and farnesoid X-activated receptor (FXR) that maintains bile acid homeostasis; previously, this feedback was known occur only in HepG2 cells. We demonstrate that KCs in 3D liver models display expression patterns consistent with an anti-inflammatory phenotype when compared to monocultures. These results highlight the distinct roles of LSECs and KCs in maintaining liver function and emphasize the need for additional mechanistic studies of NPCs in addition to hepatocytes in liver-mimetic microenvironments.