Host cyclophilin (cyp) inhibitors, such as NIM811, efficiently inhibit replication of hepatitis C virus (HCV) and have shown significant promise in recent clinical trials for the treatment of chronic HCV. It is therefore important to fully understand the mechanism of action of these therapeutic agents. Data obtained from comprehensive systems biology approaches have led to the hypothesis that the antiviral activity of cyclophilin inhibitors is mediated through impairing the cellular machinery on which HCV relies to traffic cofactors necessary for formation of the replication complex. Indeed, our results demonstrate when cyclophilins are inhibited by NIM811, lipid and protein trafficking within the VLDL pathway is impaired. Following treatment of replicon or HCV infected cells with NIM811, intracellular lipid droplets (LD) more than double in size and decrease in number. Changes in the LDs in response to cyclophilin inhibition are dependent upon expression of viral proteins. Additionally, in cells treated with NIM811, apoB accumulates in a crescent or ring shaped structure surrounding the enlarged LDs and is no longer secreted. Silencing of cypA or cyp40 using siRNA had a similar effect on LD size and apoB localization as compound treatment, suggesting these cyclophilins may play an important role in lipid and apoB trafficking. Interestingly, the decrease in apoB secretion correlates with a decrease in release of viral particles in HCV infected cells. Altogether, these results add a new level of complexity to the mechanism of action of cyclophilin inhibition, and suggest the role for cyclophilins in the virus life cycle extends beyond replication to virus release.