Coxsackievirus B3 (CVB3), an enterovirus (EV) in the family of Picornaviridae, is a global human pathogen for which effective antiviral treatments and vaccines are lacking. Previous research demonstrated that EV-D68 downregulated the membrane fusion protein SNAP47 (synaptosome associated protein 47) and SNAP47 promoted EV-D68 replication via regulating autophagy. In the current study, we investigated the interplay between CVB3 and cellular SNAP47 using HEK293T/HeLa cell models. We showed that, upon CVB3 infection, protein levels of SNAP47 decreased independent of the activity of virus-encoded proteinase 3C. We further demonstrated that the depletion of SNAP47 inhibited CVB3 infection, indicating a pro-viral function of SNAP47. Moreover, we found that SNAP47 co-localizes with the autophagy-related protein ATG14 on the cellular membrane fractions together with viral capsid protein VP1, and expression of SNAP47 or ATG14 enhanced VP1 conjugation. Finally, we revealed that disulfide interactions had an important role in strengthening VP1 conjugation. Collectively, our study elucidated a mechanism by which SNAP47 and ATG14 promoted CVB3 propagation through facilitating viral capsid assembly.