Invention of subgenomic HCV replicon a few years ago and recent success of in vitro production of infectious HCV have improved our knowledge of the HCV life cycle, replication, pathogenesis, and screening of anti-HCV therapeutics. However, the highly genotype-dependent nature of the in vitro HCV production system has limited its potential for HCV research. In this study, we constructed a recombinant DNA-based HCV system that contained EF-1alpha promoter-driven HCV genotype 1b with HCV E1/E2 deleted and replaced by GFP. We co-transfected this recombinant cDNA with HCV E1/E2 or VSV-G expression plasmid into 293T cells, and we showed HCV protein expression and processing and demonstrated production of HCV-like particles in culture supernatant of co-transfected cells. These results support potential use of this system for studies on expression and processing of the HCV polyprotein and assembly and release of HCV-like particles.