The nonstructural protein 5A (NS5A) of the hepatitis C virus (HCV) has been shown to interact with a variety of cellular proteins and implicated in the regulation of cell growth, interferon resistance, and other cellular signaling pathways, but the role of NS5A in HCV pathogenesis has not been firmly established. To further characterize this multifunctional protein, we instigated the studies of the subcellular localization of NS5A in a hepatoma cell line. NS5A was localized to the perinuclear membrane structures, including the endoplasmic reticulum (ER) and the Golgi apparatus, by immunofluorescence staining and confocal microscopy. In addition, it was also associated with the surface of cytoplasmic globular structures when expressed alone or as a part of the NS3-5B polyprotein. Oil red O staining revealed that these globular structures were lipid droplets, where the HCV core protein was also localized. The association of NS5A with intracellular membrane was further confirmed by membrane flotation analysis. To determine whether NS5A interacts with any cellular lipid-binding protein, we performed yeast two-hybrid screening in both HepG2 and human liver cDNA libraries. Apolipoprotein A1 (apoA1), one of the protein components of high-density lipoprotein (HDL) particles, was identified by two independent screening processes. The interaction between NS5A and apoA1 was confirmed by both in vitro pull-down and in vivo coimmunoprecipitation experiments. Immunofluorescence staining revealed a significant colocalization of NS5A and apoA1 in the Golgi apparatus. Our results established an association of NS5A with lipid droplets and apoA1, suggesting that NS5A, together with the core protein, may play a role in the pathogenesis of the derangement of lipid metabolism, contributing to liver steatosis commonly observed in hepatitis C.