Many picornaviruses require the myristoylation of capsid proteins for viral replication. Myristoylation is a site-specific lipidation to the N-terminal G residue of viral proteins, which is catalyzed by the ubiquitous eukaryotic enzyme N-myristoyltransferase (NMT) by allocating the myristoyl group to the N-terminal G residue. IMP-1088 and DDD85646 are two inhibitors that can deprive NMT biological functions. Whether Senecavirus A (SVA) uses NMT to modify VP0 and regulate viral replication remains unclear. Here, we found that NMT inhibitors could inhibit SVA replication. NMT1 knock-out in BHK-21 cells significantly suppressed viral replication. In contrast, the overexpression of NMT1 in BHK-21 cells benefited viral replication. These results indicated that VP0 is a potential NMT1 substrate. Moreover, we found that the myristoylation of SVA VP0 was correlated to the subcellular distribution of this protein in the cytoplasm. Further, we evaluated which residues at the N-terminus of VP0 are essential for viral replication. The substitution of N-terminal G residue, the myristoylation site of VP0, produced a nonviable virus. The T residue at the fifth position of the substrates facilitates the binding of the substrates to NMT. And our results showed that the T residue at the fifth position of VP0 played a positive role in SVA replication. Taken together, we demonstrated that SVA VP0 myristoylation plays an essential role in SVA replication.