Verticillium wilt (VW), induced by the soil-borne fungus Verticillium dahliae (Vd), poses a substantial threat to a diverse array of plant species.  Employing molecular breeding technology for the development of cotton varieties with heightened resistance to VW stands out as one of the most efficacious protective measures.  In this study, we successfully generated two stable transgenic lines of cotton (Gossypium hirsutum L.), VdThit-RNAi-1 and VdThit-RNAi-2, using host-induced gene silencing (HIGS) technology to introduce double-stranded RNA (dsRNA) targeting the thiamine transporter protein gene (VdThit).  Southern blot analysis confirmed the presence of a single-copy insertion in each line.  Microscopic examination showed marked reductions in the colonization and spread of Vd-mCherry in the roots of VdThit-RNAi cotton compared to wild type (WT).  The corresponding disease index and fungal biomass of VdThit-RNAi-1/2 also exhibited significant reductions.  Real-time quantitative PCR (qRT-PCR) analysis demonstrated a substantial inhibition of VdThit expression following prolonged inoculation of VdThit-RNAi cotton.  Small RNA sequencing (sRNA-Seq) analysis revealed the generation of a substantial number of VdThit-specific siRNAs in the VdThit-RNAi transgenic lines.  Additionally, the silencing of VdThit by the siVdThit produced by VdThit-RNAi-1/2 resulted in the elevated expression of multiple genes involved in the thiamine biosynthesis pathway in Vd.  Under field conditions, VdThit-RNAi transgenic cotton exhibited significantly enhanced disease resistance and yield compared with WT.  In summary, our findings underscore the efficacy of HIGS targeting VdThit in restraining the infection and spread of Vd in cotton, thereby potentially enabling the development of cotton breeding as a promising strategy for managing VW.