Microgravity (µg) during spaceflight affects cellular and molecular functions of both human cells and microbial pathogens, influencing viral replication and the host immune system. This study aimed to investigate the effects of simulated µg on Herpes Simplex Virus-1 (HSV-1) replication, host pro-inflammatory cytokine, and human endogenous retrovirus (HERV) activation in human neuroblastoma SH-SY5Y cells. Our results show that µg has a negative impact on HSV-1 replication, leading to significantly reduced viral titers and lower expression levels of HSV-1 early genes (ICP0, ICP4, and ICP27) compared to 1 gravity (1 g) conditions. Interestingly, despite lower viral titers and HSV-1 gene expressions under µg condition, we observed higher levels of HERVs and pro-inflammatory cytokine gene expression. In addition, there was a significant correlation between HSV-1 immediate-early genes with HERVs and pro-inflammatory cytokine gene expression, with stronger correlations observed under µg conditions. Taken together, µg reduces HSV-1 replication and increases host pro-inflammatory and HERVs gene expression, which demands further investigation for human health protection in space.