Hepatitis C virus (HCV) genotype 3a is widespread worldwide, but no replication system exists for its study. We describe a subgenomic replicon system for HCV genotype 3a. We determined the consensus sequence of an HCV genome isolated from a patient, and constructed a subgenomic replicon using this clone. The replicon was transfected into HuH-7 cells and RNA replication was confirmed. We identified cell culture-adaptive mutations that increased colony formation multiple-fold. We have therefore established a genotype 3a replicon system that can be used to study this HCV genotype.