Lentiviral vectors are widely used for stable gene delivery, but their transduction efficiency can be limited by suboptimal experimental conditions. Here, we investigated the role of oxygen concentration and hypoxia-inducible factor 1 (HIF-1) signaling in lentiviral packaging and transduction. We found that packaging lentivirus under hypoxic conditions (10% O₂) significantly increased viral titers and transduction efficiency by approximately 10%. However, hypoxic conditions during viral entry impaired infection efficiency, likely due to HIF-1α-mediated cellular protective mechanisms. Pretreatment of cells with the HIF-1 inhibitor PX-478 reversed this effect, enhancing viral entry and genome integration in a dose-dependent manner. Combining hypoxic virus packaging with PX-478 pretreatment synergistically improved transduction efficiency by 20%. These findings suggest that HIF-1 inhibition and controlled hypoxia significantly enhance lentiviral transduction efficiency, establishing a versatile strategy with broad applicability across viral vector-dependent biomedical applications.