Zika virus (ZIKV) is an important human pathogen associated with severe neurological disorders. Ubiquitination of viral proteins has diverse roles in viral life cycle and pathogenesis. Here, we found that perturbation of ubiquitin-proteasome system significantly suppressed production of infectious viral particles. Moreover, we demonstrated that ZIKV precursor membrane (prM) protein underwent ubiquitination and proteasomal degradation. Furthermore, we showed that co-expression of E protein with ubiquitination-deficient prM-6 K/6R mutant protein did not affect translocation of viral proteins into endoplasmic reticulum and trans-Golgi networks. Intriguingly, the co-expression of E and prM-6 K/6R mutant proteins led to formation of relatively aggregated viral protein complexes and resulted in diminishing secretion of viral proteins as compared to wild-type prM. Collectively, these results suggest that ubiquitinated ZIKV prM protein contributes to the release of viral proteins and provide a new insight into the mechanism involved in ZIKV replication biology.Copyright © 2020. Published by Elsevier B.V.