Verticillium wilt is a devastating disease of cotton, which is caused by the soil-borne fungus Verticillium dahliae (V. dahliae). Although previous studies have identified some genes or biological processes involved in the interaction between cotton and V. dahliae, its underlying molecular mechanism remains unclear, especially in G. hirsutum. In the present study, we obtained an overview of transcriptome characteristics of resistant upland cotton (G. hirsutum) after V. dahliae infection at 24 h post-inoculation (hpi) via a high-throughput RNA-sequencing technique. A total of 4,794 differentially expressed genes (DEGs) were identified, including 820 up-regulated genes and 3,974 down-regulated genes. The enrichment analysis showed that several important processes were induced upon V. dahliae infection, such as plant hormone signal transduction, plant-pathogen interaction, phenylpropanoid-related and ubiquitin-mediated signals. Moreover, we investigated some key regulatory gene families involved in the defense response, such as receptor-like protein kinases (RLKs), WRKY transcription factors and cytochrome P450 (CYPs), via virus-induced gene silencing (VIGS). GhSKIP35, a partner of SKP1 protein, was involved in ubiquitin-mediated signal. Over-expression of GhSKIP35 in Arabidopsis improved its tolerance to Verticillium wilt in transgenic plants. Collectively, global transcriptome analysis and functional gene characterization provided significant insights into the molecular mechanisms of G. hirsutum-V. dahliae interaction and offered a number of candidate genes as potential sources for breeding wilt-tolerance in cotton.