Abstract Verticillium wilt (VW) caused by Verticillium dahliae (V. dahliae) is one of the most destructive diseases in cotton (Gossypium spp.). Histone acetylation plays critical roles in plant development and adaptive responses to biotic and abiotic stresses. However, the relevance of histone acetylation in cotton VW resistance remains largely unclear. Here, we identified histone deacetylase 5 (GhHDA5) from upland cotton (Gossypium hirsutum L.), as a negative regulator of VW resistance. GhHDA5 expression was responsive to V. dahliae infection. Silencing GhHDA5 in upland cotton led to improved resistance to V. dahliae, while heterologous expression of GhHDA5 in Arabidopsis (Arabidopsis thaliana) compromised V. dahliae tolerance. GhHDA5 repressed the expression of several lignin biosynthesis-related genes, such as 4-coumarate:CoA ligase gene Gh4CL3 and ferulate 5-hydroxylase gene GhF5H, through reducing the acetylation level of histone H3 lysine 9 and 14 (H3K9K14ac) at their promoter regions, thereby resulting in an increased deposition of lignin, especially S monomers, in the GhHDA5-silenced cotton plants. The silencing of GhF5H impaired cotton VW tolerance. Additionally, the silencing of GhHDA5 also promoted the production of reactive oxygen species (ROS), elevated the expression of several pathogenesis-related genes (PRs), and altered the content and signaling of the phytohormones salicylic acid (SA), jasmonic acid (JA), and strigolactones (SLs) after V. dahliae infection. Taken together, our findings suggest that GhHDA5 negatively regulates cotton VW resistance through modulating disease-induced lignification and the ROS- and phytohormone-mediated defense response.