Enterovirus 71 (EV71) causes hand, foot and mouth disease (HFMD), a mild and self-limited illness that is sometimes associated with severe neurological complications among young children and immunosuppressed patients. The aim of this study was to better identify the viral determinants of EV71 dissemination in humans, and to propose new therapeutic approaches against EV71 infection. We demonstrated that the presence of the positively charged arginine in the VP1-97 residue of EV71 capsid, which was previously identified during viral intra-host adaptation within an immunocompromised host, enabled the virus to bind to the heparan sulfate (HS) attachment receptor without modifying its dependence on the main entry receptor (SCARB2). HS-dependent variants displayed modified viral tropism in vitro and ex vivo and ι-carrageenan, a highly sulfated compound blocked their replication in vitro. These findings propose that the acquired dependency of the mutated variant for HS attachment receptor conferred the virus the ability to disseminate within the immunocompromised host and open the door to new approaches to treat EV71 infection.