In recent years, CVA6 has become a new pathogen of Hand, Foot, and Mouth Disease (HFMD), associated with crusted lesions, a rash affecting the arms, trunk, and legs, nail loss. In order to dig out the unclear pathogenesis of CVA6-associated HFMD, we established a reverse genetic system for CVA6 and determined the virulence and the proliferation of the rescued virus. In this study, the CVA6 viral cDNA was divided into two segments, which were ligated through the BclI restriction site. The constructed plasmid was designated as pWSK29-CVA6-89R identified by enzyme digestion and the whole cDNA sequencing. The rescued virus CVA6-89R was infected in ICR mice, showing high pathogenicity. This study established a reverse genetic system for CVA6.