Zika virus (ZIKV) is an emerging mosquito-borne flavivirus that rapidly became a major medical concern worldwide. We have recently reported that a high glucose level decreases the rate of Zika virus (ZIKV) replication with an impact on human kidney HK-2 cell survival. However, the mechanisms by which cells cultured in a high glucose medium inhibit ZIKV growth remain unclear. Viperin belongs to interferon-stimulated genes (ISG) and its expression is highly up-regulated upon viral infection, leading to antiviral activity against a variety of viruses, including flaviviruses. As such, viperin has been shown to be a major actor involved in the innate immune response against Zika virus (ZIKV). Our present study aims to further characterize the involvement of viperin in ZIKV growth inhibition under high glucose concentration (HK-2HGC). We show for the first time that endogenous viperin is over-expressed in HK-2 cells cultured under high glucose concentration (HK-2HGC), which is associated with ZIKV growth inhibition. Viperin knockdown in HK-2HGC rescues ZIKV growth. In addition, our results emphasize that up-regulated viperin in HK-2HGC leads to ZIKV growth inhibition through the stimulation of IFN-β production. In summary, our work provides new insights into the ZIKV growth inhibition mechanism observed in HK-2 cells cultured in a high glucose environment.