MicroRNAs play essential roles during defense responses in plants, yet their roles have not been widely functionally validated in cotton response to Verticillium dahliae. Here, we employed transgenic technology, virus induced gene silencing technology, as well as various cytological and molecular tools to investigate the function of miR398b and its target genes in cotton response to V. dahliae. Transcript levels of miR398b were down-regulated by V. dahliae infection and miR398b overexpression in cotton made the plants more susceptible to V. dahliae. The results suggest that miR398b negatively regulates cotton resistance to V. dahliae via two possibilities. One is that miR398b may repress some CC-NBS-LRR genes during transcriptional or translational processes, thereby interfering with defense responses of cotton to V. dahliae and causing increased susceptibility of cotton to V. dahliae. Another possibility is that miR398b may guide the cleavage of the mRNAs of GhCSD1, GhCSD2 and GhCCS, which are important in the regulation of ROS homeostasis, thereby leading to excessive ROS accumulation in miR398b-overexpressing plants during V. dahliae infection. These studies illuminate the conserved and novel roles of miR398b during the cotton-V. dahliae interaction, which yields insights into new strategies to improve resistance to V. dahliae in cotton breeding programs