CGI58 (Comparative Gene Identifier 58) is considered a key regulator of lipolysis since the disruption of CGI58 in Arabidopsis results in the hyper-accumulation of lipid droplets in vegetative tissues. However, the molecular mechanism underlying this process has not been well studied. To address this, Arabidopsis lines stably expressing RNAi against transcripts encoding one or both of the CGI58 isoforms were generated and, using RT-PCR, specific alterations in CGI58 gene expression were shown to be achieved. Additionally, split-ubiquitin two-hybrid and bimolecular fluorescence complementation assays indicated that CGI58 interacts, albeit weakly, with PXA1, a peroxisomal transport protein responsible for the uptake of various lipid metabolites. Finally, results from mutational analyses of PXA1 revealed that the protein’s peroxisomal membrane targeting information is located within its N-terminal 180 amino acids. Taken together, these results have important implications for understanding the coordinated roles of CGI-58 and PXA1 in regulating lipid homeostasis in plants.