Successful replication of flaviviruses such as Zika virus (ZIKV) requires a highly coordinated replication cycle involving various interactions with host enzymes. We show herein that the NS3 protein of ZIKV is acetylated in human cells during protein overexpression and authentic infection. The acetylated state of the ZIKV NS3, specifically at residue K389 within its helicase domain, was found to regulate its RNA binding and unwinding capacity. The dynamic regulation of the acetylated state of ZIKV NS3 was required to achieve efficient viral replication in several cell types. Our study provides molecular insight into host-mediated post-translational regulation of the ZIKV NS3 protein, revealing a potential new therapeutic avenue for antiviral drug development. Lysine acetyltransferases are a class of enzymes that reversibly transfer an acetyl group from the cofactor acetyl-CoA onto the E-amino group of a lysine residue within a target protein. The addition of the acetyl group neutralizes the positive charge of the lysine residue, and can have myriad downstream consequences for protein function. Herein, we took an RNAi approach to identify the host acetyltransferase responsible for lysine acetylation of the ZIKV NS3 K389 residue. Our efforts discovered that KAT5g, a hitherto uncharacterized, replication complex-localized isoform of the host lysine acetyltransferase 5 (KAT5), is required for NS3 K389 acetylation and regulation of ZIKV replication. The flaviviruses are a family of vector-borne pathogens responsible for significant morbidity and mortality across the global population [184]. The mosquito-borne flaviviruses, such as ZIKV, WNV, DENV, and YFV, are the most common culprits for causing flavivirus-associated diseases in humans. Both ZIKV and WNV present with neurological symptoms during human infection given their neuronal tropism. In terms of neuropathology, ZIKV most often presents in newborn babies after crossing the trans-placental barrier and infecting neuro-progenitor cells during fetal development, while WNV infection is more commonly associated with meningoencephalitis in immuno-compromised adults [185]. Here, we show that robust acetylation of the NS3 helicase is conserved in WNV. Moreover, we found that mutation of K389 reduces acetylation of WNV NS3. In turn, we tested whether KAT5 was the enzyme responsible for acetylation of WNV NS3 and found that proviral acetylation of NS3 by KAT5/KAT5g is a conserved mechanism controlling both ZIKV and WNV replication, providing the potential for an antiviral therapy capable of restricting multiple flaviviruses.