PROJECT SUMMARY Peroxisomes are ubiquitous membrane bound organelles that play a major role in the regulation of lipid metabolism. In the liver, peroxisomes contain enzymes involved in essential processes such as the detoxification of reactive oxygen species (ROS), the oxidization of fatty acids, and synthesis of bile acids, the body’s natural detergents and potent signaling molecules. The generation of peroxisomes is driven by the peroxin (herein PEX) protein family. However, the role of PEX proteins in coordinating inter-organelle crosstalk has not been explored. Together, the mitochondria, endoplasmic reticulum (ER), and peroxisomes maintain lipid homeostasis and regulate lipid metabolism within hepatocytes. How organelles involved in lipid metabolism, such as the mitochondria and the ER, respond to changes in peroxisomal function is not well understood. Here, we describe the role of two PEX family proteins, PEX6 and PEX14, in hepatic lipid metabolism. Using a panel of over 100 inbred strains of genetically diverse mice, we found that PEX6 and PEX14 protein levels positively correlated with lipid levels in the liver. We therefore hypothesized that PEX6 and PEX14 may have specific roles in regulating lipid and bile acid metabolism through the import of critical components into peroxisomes. To test this, we generated AAV-CRISPR to disrupt Pex6 and Pex14 in the liver of adult mice. Mice injected with Pex6- or Pex14-CRISPR did not put on/gain body weight when fed a standard rodent chow diet, however there was no difference in their lean mass, plasma ALT/AST, or liver histology, compared to control mice, suggesting they were not sick. In addition, we observed a significant increase of hepatic bile acids in both Pex14-CRISPR and Pex6-CRISPR animals indicating dysregulation of BA synthesis within peroxisomes. Further, lack of either PEX6 or PEX14 resulted in specific increases in ROS metabolism and fatty acid oxidation. Together, these data suggest that loss of hepatic PEX14 or PEX6 augment peroxisome activity. Here, we aim to investigate how these peroxisomal changes may impact the morphology and function of other organelles involved in lipid metabolism, specifically the mitochondria and the ER. Preliminary results suggest that in mice lacking PEX14 or PEX6, mitochondrial respiration is significantly increased, and the ER is expanded. Further, we aim to investigate whether lipid accumulation, mimicking metabolic disease states, impact these observations. The proposed studies will describe one example of how the loss of two PEX proteins affects lipid homeostasis and inter-organelle crosstalk, thus advancing our understanding of lipid metabolism in healthy and disease states.