Verticillium dahliae is a destructive soil-borne fungal pathogen that causes Verticillium wilt in a wide range of economically important crops, yet effective and durable control strategies remain limited. Host-induced gene silencing (HIGS) has emerged as a promising approach for suppressing fungal virulence genes; however, the influence of double-stranded RNA (dsRNA) architecture on RNA interference (RNAi) efficacy remains poorly understood. In this study, we systematically engineered four hairpin RNA (hpRNA) constructs targeting a novel V. dahliae gene to investigate how structural modifications affect dsRNA stability, processing, and small interfering RNA (siRNA) biogenesis. The constructs included a conventional hpRNA, an hpRNA with altered processing (hpRNA[G:U]), a stabilized hpRNA (LedRNA), and an hpRNA designed to alter siRNA size-class production. These constructs were evaluated using both transient expression assays and stable transgenic Nicotiana benthamiana plants. Northern blot analyses revealed clear construct-dependent differences in full-length dsRNA accumulation and processing, demonstrating that hpRNA architecture strongly influences dsRNA stability and siRNA production. Moreover, stable transgenic plants expressing these constructs exhibited enhanced resistance to Verticillium wilt. Collectively, these findings provide insights into the role of hpRNA design in RNAi efficiency and support the development of more effective RNAi-based strategies for the control of V. dahliae and other economically important fungal pathogens.