<p>Single-cycle viruses, which lack essential genes for virus replication, demonstrate high protective efficacy and safety, making them promising candidates for commercial vaccines. However, their lack of replication capacity presents a difficulty for large-scale vaccine production. In this study, we aimed to enhance viral replication by utilizing drugs targeting microtubule dynamics, which play a crucial role in maintaining cell structure, transporting materials, and facilitating the movement of viral particles within the host cell during virus infection. To identify optimal drug concentrations and assess viral replication capacity, we constructed the single-cycle viral hemorrhagic septicemia virus rVHSV-A-MetLuc-GΔTM, which contains a luciferase reporter gene. Using this construct, both drugs enhanced the production of single-cycle viruses in fish cells at a concentration of 100 nM without causing cytotoxicity. Furthermore, we identified the mechanism by which viral replication is increased, which involves upregulation of the c-Fos and c-Jun genes. This indicates that the transcription factor AP-1 plays a key role in this process. This study shows that appropriate treatment with colchicine and paclitaxel, which influence microtubule dynamics, can enhance the replication capacity of single-cycle viruses. This presents opportunities to increase the cost-effectiveness of single-cycle and attenuated virus-based vaccines for industrial use.</p>