Zika virus (ZIKV) infection in humans has been linked to serious birth defects, including abnormalities of the brain and eyes. Here, we used two lines of primary human glial cells from the brain and found both are susceptible to ZIKV infection. Viral RNA abundance increased from 6 to 36 h post-infection (hpi). Both cells showed cytopathic effects with cell shedding, suspension and death at 48 hpi. We found increased mRNA levels of IL-6, IL-1b, CXCL10 (IP-10), IL-8 and CCL2 in ZIKV-infected microglial cells, as well as those of TNF-a in astrocytes. Elevated levels of CXCL10 protein were also observed in microglial cells at 48 hpi. These results suggested that glial cells may directly contribute to neuroinflammation by releasing pro-inflammatory cytokines. ZIKV infection can also activate ISGs, as evidenced by increased mRNA levels of IFN-b, OAS1, Mx1 and IFITM1 in infected microglia at 48 hpi. In addition, ZIKV can induce levels of caspase-1 and caspase-3 expression in microglia and astrocytes, respectively. These findings suggest that activation of glial cells contributes to neuroinflammation and induces distinct cell death pathways in different glial cells. Finally, we examined the antiviral effect of azithromycin in infected glial cells and found a dose-dependent effect (0, 10 and 20 uM) on viral reduction. Azithromycin treatment significantly decreased gene expression of TNF-a. Moreover, the antiviral effect of azithromycin was confirmed in mouse models of ZIKV infection. Collectively, our study demonstrated that ZIKV can infect primary human glial cells, leading to inflammatory responses and cell death. Azithromycin treatment inhibits viral load in glial cells and alleviate neuroinflammation during ZIKV infection.