ABSTRACTSalinity stress is a major abiotic stress affecting the productivity and fiber quality of cotton. Although reactive oxygen species (ROS) play critical roles in plant stress responses, their complex molecular regulatory mechanism under salinity stress is largely unknown in cotton, especially microRNA (miRNA)-mediated regulation of superoxide dismutase gene expression. Here, we report that a cotton iron superoxide dismutase gene GhFSD1 and the cotton miRNA ghr-miR414c work together in response to salinity stress. The miRNA ghr-miR414c targets the coding sequence region of GhFSD1, inhibiting expression of transcripts of this antioxidase gene, which represents the first line of defense against stress-induced ROS. Expression of GhFSD1 was induced by salinity stress. Under salinity stress, ghr-miR414c showed expression patterns opposite to those of GhFSD1. Ectopic expression of GhFSD1 in Arabidopsis conferred salinity stress tolerance by improving primary root growth and biomass, whereas Arabidopsis constitutively expressing ghr-miR414c showed hypersensitivity to salinity stress. Silencing GhFSD1 in cotton caused an excessive hypersensitive phenotype to salinity stress, whereas overexpressing miR414c decreased the expression of GhFSD1 and increased sensitivity to salinity stress, yielding a phenotype similar to that of GhFSD1-silenced cotton. Taken together, our results demonstrated that ghr-miR414c was involved in regulation of plant response to salinity stress by targeting GhFSD1 transcripts. This study provides a new strategy and method for plant breeding in order to improve plant salinity tolerance.