Verticillium wilt (VW), induced by the soil-borne fungus Verticillium dahliae, represents a significant threat to global cotton production. Phenylalanine ammonia-lyase (PAL) is an essential enzyme in lignin metabolism that helps plants defend themselves against pathogenic fungal. Nonetheless, its role in imparting resistance to V. dahliae in cotton required further investigation. This study identified the GhPAL9 (GH_D04G1247) as a crucial gene in cotton resistance to V. dahliae via RNA-seq analysis, demonstrating significant upregulation in the resistant variety Xinluzao84. Bioinformatics analysis revealed the conserved evolutionary relationship of GhPAL9 with PAL homologs across various species and highlighted stress-responsive cis-elements in its promoter region. The expression of GhPAL9 was rapidly activated in roots, stems, and leaves following infection with V. dahliae, peaking between 2 and 8 h post inoculation (hpi). Silencing GhPAL9 through virus-induced gene silencing (VIGS) technology intensified disease symptoms, elevated relative fungal biomass, and diminished lignin accumulation, thereby affirming its function in cotton resistance to V. dahliae. The overexpression of GhPAL9 in Arabidopsis improved resistance to V. dahliae, and the OE-GhPAL9 transgenic lines demonstrated reduced disease severity and diminished relative fungal biomass. The results gave us new information about how VW resistance at the molecular level, which established that GhPAL9 acted as a positive regulator to increase resistance to VW via lignin accumulation.