Hepatitis C virus (HCV) infection is a major worldwide health problem. The envelope glycoproteins are the major components of viral particles. Here we developed a trans-complementation system that allows the production of infectious HCV particles in whose genome the regions encoding envelope proteins are deleted (HCVΔE). The lack of envelope proteins could be efficiently complemented by the expression of homologous envelope proteins in trans. HCVΔE production could be enhanced significantly by previously described adaptive mutations in NS3 and NS5A. Moreover, HCVΔE could be propagated and passaged in packaging cells stably expressing HCV envelope proteins, resulting in only single-round infection in wild-type cells. Interestingly, we found that vesicular stomatitis virus (VSV) glycoproteins could efficiently rescue the production of HCV lacking endogenous envelope proteins, which no longer required apolipoprotein E for virus production. VSV glycoprotein-mediated viral entry could allow for the bypass of the natural HCV entry process and the delivery of HCV replicon RNA into HCV receptor-deficient cells. Our development provides a new tool for the production of single-cycle infectious HCV particles, which should be useful for studying individual steps of the HCV life cycle and may also provide a new strategy for HCV vaccine development.