Picornaviruses have evolved to hijack host cellular machinery, including the autophagic pathway. However, the mechanisms remain largely unclear. We use coxsackievirus B3 (CVB3) as a model organism to explore the possible role of picornavirus subversion of the autophagic pathway in viral infection. Our in vivo and in vitro experiments demonstrate that CVB3 infection causes a significant, albeit incomplete, inhibition of autophagic flux by limiting the fusion of autophagosomes with lysosomes and/or late endosomes. Furthermore, we show that CVB3 specifically targets SNARE protein SNAP29 and adaptor protein PLEKHM1, two critical proteins known to regulate autophagosome fusion, for cleavage through the catalytic activity of viral proteinase 3C, ultimately impairing the formation of SNARE complexes. Finally, we demonstrate that loss of SNAP29/PLEKHM1 inhibits autophagic flux, resulting in increased viral replication. Collectively, our study reveals a mechanism that supports an emerging model whereby CVB3 hijacks the autophagic machinery to facilitate its own propagation. Display omitted •CVB3 infection inhibits autophagosome-lysosome/late endosome fusion•CVB3 limits autophagic flux via cleavage of SNAP29 and PLEKHM1 by proteinase 3C•Knockdown of SNAP29 and/or PLEKHM1 facilitates viral replication Mohamud et al. report that coxsackievirus limits autophagosome fusion with lysosomes and/or late endosomes through viral proteinase 3C-mediated cleavage of SNAP29 and PLEKHM1. This leads to the accumulation of autophagosomes that favor viral replication by providing additional membrane scaffolds.